Synthetic linear apelin mimetics for the treatment of heart failure

ABSTRACT

The invention provides a synthetic polypeptide of Formula I′ (SEQ ID NO: 1):
 
X1-X2-X3-R—X5-X6-X7-X8-X9-X10-X11-X12-X13  I
         or an amide, an ester or a salt thereof, wherein X1, X2, X3, X5, X6, X7, X8, X9, X10, X11, X12 and X13 are defined herein. The polypeptides are agonist of the APJ receptor. The invention also relates to a method for manufacturing the polypeptides of the invention, and its therapeutic uses such as treatment or prevention of acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral arterial disease, cerebrovascular accidents, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn) and preeclampsia. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

This application claims benefit under 35 U.S.C. §119(e) of USProvisional Application No. 61/728,395 filed Nov. 20, 2012; U.S.Provisional Application No. 61/781,397, filed on Mar. 14, 2013; thecontents of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 21, 2014, isnamed PAT055326-US-NP_SL.txt and is 68,146 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel compositions comprising modified peptideand polypeptide sequences designed to treat cardiovascular disease insubjects to whom they are administered, and which exhibit greaterresistance to degradation, and equivalent or greater bioactivity thantheir wild type counterparts. The invention also relates to methods ofmaking said compositions and using said compositions as pharmaceuticallyactive agents to treat cardiovascular disease.

BACKGROUND OF THE INVENTION

The incidence of heart failure in the Western world is approximately1/100 adults after 65 yrs of age. The most common pathology is a chronicdeficit in cardiac contractility and, thereby, cardiac output, i.e., theeffective volume of blood expelled by either ventricle of the heart overtime. Patients with chronic heart failure can have acute episodes ofdecompensation, i.e., failure of the heart to maintain adequate bloodcirculation, where cardiac contractility declines further. There are˜500K hospitalizations per year for “acute decompensated heart failure”(ADHF) in the USA alone.

Current therapies for ADHF include diuretics, vasodilators, andinotropes, which directly increase cardiac contractility. Currentintravenous inotropes (dobutamine, dopamine, milrinone, levosimendan)are used in the acute setting, despite their association with adverseevents such as arrhythmia and increased long-term mortality. Theseliabilities have prevented their application in chronic heart failure.Digoxin is an oral inotrope, but is limited by a narrow therapeuticindex, increased arrhythmogenic potential and contraindication in renalinsufficiency.

A therapy for heart failure that increases cardiac contractility withoutarrhythmogenic or mortality liabilities is urgently needed for ADHF, butcould also address the enormous unmet medical need in chronic heartfailure.

Apelin is the endogenous ligand for the previously orphanG-protein-coupled receptor (GPCR), APJ, also referred to as apelinreceptor, angiotension-like-1 receptor, angiotension II-like-1 receptor,and the like. The apelin/APJ pathway is widely expressed in thecardiovascular system and apelin has shown major beneficialcardiovascular effects in preclinical models. Acute apelinadministration in humans causes peripheral and coronary vasodilatationand increases cardiac output (Circulation. 2010; 121:1818-1827). As aresult, APJ agonism is emerging as an important therapeutic target forpatients with heart failure. Activation of the apelin receptor APJ isthought to increase cardiac contractility and provide cardioprotection,without the liabilities of current therapies. However, the nativeapelins exhibit a very short half life and duration of action in vivo.

It is therefore desirable to identify peptides and polypeptides thatmimic the function of apelin, but have increased half-life anddemonstrate equivalent or greater bioactivity than the naturallyoccurring apelin. Furthermore, it is desirable to identify apelin analogpeptides and polypeptides which exhibit increased conformationalconstraints, i.e., the ability to achieve and maintain an activeconformational state such that the peptides and polypeptides caninteract with their receptors and/or other pathway targets without theneed for additional folded or repositioning. There is a need for use ofsuch peptide and polypeptide analogs, compositions comprising saidanalogs, and methods of making and using said compositions aspharmaceutically active agents to treat diseases, such as cardiovasculardiseases.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide novel peptides andpolypeptides which are useful as APJ agonists, and which also possess atleast one of the following improvements over wild type apelin and otherknown apelin analogs: increased half-life; greater immunity todegradation upon administration and/or upon solubilization; andincreased conformational constraints, all while exhibiting the same orgreater biological activity as wild type apelin. The peptides andpolypeptides of this invention are thus particularly useful for thetreatment or prevention of cardiovascular diseases such as heartfailure, disorders and conditions associated with heart failure, anddisorders and conditions responsive to the activation of APJ receptoractivity.

In one embodiment, the peptides and polypeptides of the invention areparticularly useful for the treatment or prevention of a disorder orcondition associated with heart failure, or a disorder responsive to theactivation (or agonism) of the APJ receptor activity. In anotherembodiment, the peptides and polypeptides of the invention are useful inthe treatment of acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

The invention pertains to the peptides and polypeptides, pharmaceuticalcompositions, and methods of manufacture and use thereof, as describedherein. Examples of peptides and polypeptides of the invention includethe peptides and polypeptides according to any one of Formulae I to IV,or an amide, an ester or a salt thereof, as well as any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

The invention therefore provides a peptide or a polypeptide formula (I)SEQ ID NO: 1):X1-X2-X3-R—X5-X6-X7-X8-X9-X10-X11-X12-X13  Iwherein:X1 is the N-terminus of the polypeptide and is either absent or pE;X2 is R or r;X3 is P or 4-PhP;X5 is L, Cha, D-L, F, Y, Y(Bzl), 3,4-Cl2-F or Nal;X6 is a D-amino acid, S or A;X7 is a D-amino acid, L, H or Aib; and at least one of X6 and X7 isD-amino acid or Aib;X8 is K, k, Q or E;X9 is G or D;X10 is P or pipecolic acid;X11 is D-Nle, Nle, f or D-Nva;X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 andX13 is a D-amino acid;wherein:Nle is L-norleucine;D-Nle is D-norleucine;Nal is L-(naphthyl)alanine;D-Nva is D-norvaline;Aib is α-aminoisobutyric acid;Cha is (S)-ρ-cyclohexylalanine;D-Tic is D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;pE is L-pyroglutamic acid;3,4-Cl2-F is (S)-3,4-dichlorophenylalanine;Y is L-tyrosine; andY(Bzl) is L-benzyl-tyrosine;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

As further explained herein, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D,” the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the L-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe D-amino acid.

Any of the above-listed amino acid residues of Formula I, or its relatedformulae described herein, e.g., Formulae I to IV, may be substituted ina conservative fashion, provided the peptide or polypeptide of theinvention still retains functional activity and structural properties(e.g., half-life extension, protection from degradation, conformationalconstraint). Principle and examples of permissible conservative aminoacid substitutions are further explained herein.

The polypeptides of the invention, by activating APJ receptor, haveutility in the treatment of acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

In a preferred embodiment the polypeptides of the invention are usefulin the treatment of acute decompensated heart failure (ADHF).

In another embodiment, the invention pertains to a method for treatingdisorder or disease responsive to the activation of the APJ receptor, ina subject in need of such treatment, comprising: administering to thesubject an effective amount of a polypeptide according to anyone ofFormulae I to IV, or an amide, an ester of a salt thereof, such that thedisorder or disease responsive to the activation of the APJ receptor inthe subject is treated.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions, comprising a polypeptide according to anyone of Formulae Ito IV, or an amide, an ester or salt thereof, and one or morepharmaceutically acceptable carriers.

In still another embodiment, the invention pertains to combinationsincluding, a polypeptide according to anyone of Formulae I to IV, or anamide, an ester or a salt thereof, and pharmaceutical combinations ofone or more therapeutically active agents.

In another embodiment, the invention pertains to a method for activationof the APJ receptor in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of apolypeptide according to anyone of Formulae I to IV, or an amide, anester or a salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of interpreting this specification, the followingdefinitions will apply unless specified otherwise and wheneverappropriate, terms used in the singular will also include the plural andvice versa.

As used herein, “disorders or diseases responsive to the modulation ofthe APJ receptor,” “disorders and conditions responsive to themodulation of the APJ,” “disorders and conditions responsive to themodulation of APJ receptor activity,” “disorders responsive to theactivation (or agonism) of the APJ receptor activity,” and like termsinclude acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

As used herein, “Activation of APJ receptor activity,” or “Activation ofthe APJ receptor,” refers to an increase in the APJ receptor activity.The activation of the APJ receptor activity is also referred to as“agonism” of the APJ receptor, e.g., by administration of the peptidesand polypeptides of the invention.

As used herein, the terms “polypeptide” and “peptide” are usedinterchangeably to refer to two or more amino acids linked together.Except for the abbreviations for the uncommon or unatural amino acidsset forth in Table 1 below, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D”, the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the D-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe L-amino acid. Groups or strings or amino acid abbreviations are usedto represent peptides. Peptides are indicated with the N-terminus on theleft and the sequence is written from the N-terminus to the C-terminus.

Peptides of the invention contain non-natural amino acids (i.e.,compounds that do not occur in nature) and other amino acid analogs asare known in the art may alternatively be employed.

Certain non-natural amino acids can be introduced by the technologydescribed in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wangand Schultz, Science 301:964-967, 2003; Wang et al., Science292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S.Pat. No. 7,083,970. Briefly, some of these expression systems involvesite-directed mutagenesis to introduce a nonsense codon, such as anamber TAG, into the open reading frame encoding a polypeptide of theinvention. Such expression vectors are then introduced into a host thatcan utilize a tRNA specific for the introduced nonsense codon andcharged with the non-natural amino acid of choice. Particularnon-natural amino acids that are beneficial for purpose of conjugatingmoieties to the polypeptides of the invention include those withacetylene and azido side chains.

One or more of the natural or un-natural amino acids in a peptide of theinvention may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Saidmodifications may be done in a site-specific or non-site-specificmanner. In a preferred embodiment, the modifications of the peptide leadto a more stable peptide (e.g., one exhibiting greater half-life invivo). These modifications may include the incorporation of additionalD-amino acids, etc. None of the modifications should substantiallyinterfere with the desired biological activity of the peptide, but suchmodifications may confer desirable properties, e.g., enhanced biologicalactivity, on the peptide.

Said modifications enhance the biological properties of the proteins ofthe invention relative to the wild-type proteins, as well as, in somecases, serving as points of attachment for, e.g., labels and proteinhalf-life extension agents, and for purposes of affixing said variantsto the surface of a solid support.

In certain embodiments, such modifications, e.g., site-specificmodifications, are used to attach conjugates, e.g., PEG groups topolypeptides, and/or peptides of the invention, for purposes of, e.g.,extending half-life or otherwise improving the biological properties ofsaid polypeptides, and/or peptides. Said techniques are describedfurther herein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used to attach other polymers and small molecules andrecombinant protein sequences that extend half-life of the polypeptideof the invention. One such embodiment includes the attachment of fattyacids or specific albumin binding compounds to polypeptides, and/orpeptides. In other embodiments, the modifications are made at aparticular amino acid type and may be attached at one or more sites onthe polypeptides.

In other embodiments, such modifications, e.g., site-specificmodifications, are used as means of attachment for the production ofwild-type and/or variant multimers, e.g., dimers (homodimers orheterodimers) or trimers or tetramers. These multimeric proteinmolecules may additionally have groups such as PEG, sugars, and/orPEG-cholesterol conjugates attached or be fused either amino-terminallyor carboxy-terminally to other proteins such as Fc, Human Serum Albumin(HSA), etc.

In other embodiments, such site-specific modifications are used toproduce proteins, polypeptides and/or peptides wherein the position ofthe site-specifically incorporated pyrrolysine or pyrrolysine analogueor non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe)allows for controlled orientation and attachment of such proteins,polypeptides and/or peptides onto a surface of a solid support or tohave groups such as PEG, sugars and/or PEG-cholesterol conjugatesattached.

In other embodiments, such site-specific modifications are used tosite-specifically cross-link proteins, polypeptides and/or peptidesthereby forming hetero-oligomers including, but not limited to,heterodimers and heterotrimers. In other embodiments, such site-specificmodifications are used to site-specifically cross-link proteins,polypeptides and/or peptides thereby forming protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates. In other embodiments, a site specificmodification may include a branching point to allow more than one typeof molecule to be attached at a single site of a protein, polypeptide orpeptide.

In other embodiments, the modifications listed herein can be done in anon-site-specific manner and result in protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates of the invention.

In some embodiments, the present invention provides complexes whichcomprise at least one peptide or polypeptide of anyone of Formulae I-IXbound to an antibody, such as an antibody why specifically binds apeptide or polypeptide as disclosed herein.

One of ordinary skill in the art will appreciate that various amino acidsubstitutions, e.g, conservative amino acid substitutions, may be madein the sequence of any of the polypeptides described herein, withoutnecessarily decreasing its activity. As used herein, “amino acidcommonly used as a substitute thereof” includes conservativesubstitutions (i.e., substitutions with amino acids of comparablechemical characteristics). For the purposes of conservativesubstitution, the non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophanand methionine. The polar (hydrophilic), neutral amino acids includeserine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions includesubstituting an L-amino acid for its corresponding D-amino acid,substituting cysteine for homocysteine or other non natural amino acidshaving a thiol-containing side chain, substituting a lysine forhomolysine, diaminobutyric acid, diaminopropionic acid, ornithine orother non natural amino acids having an amino containing side chain, orsubstituting an alanine for norvaline or the like.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” refers to materials which are found innature and are not manipulated by man. Similarly, “non-naturallyoccurring,” “un-natural,” and the like, as used herein, refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man. When used in connection with aminoacids, the term “naturally occurring” refers to the 20 conventionalamino acids (i.e., alanine (A or Ala), cysteine (C or Cys), asparticacid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe),glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine(K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N orAsn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg),serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophanON or Trp), and tyrosine (Y or Tyr)).

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) apelin protein sequence or thesequences of the present invention. These include, but are not limitedto, modified amino acids and/or amino acid analogues that are not one ofthe 20 naturally occurring amino acids, selenocysteine, pyrrolysine(Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCTpatent publication WO2010/48582). Such non-natural amino acid residuescan be introduced by substitution of naturally occurring amino acids,and/or by insertion of non-natural amino acids into the naturallyoccurring (wild-type) Apelin protein sequence or the sequences of theinvention. The non-natural amino acid residue also can be incorporatedsuch that a desired functionality is imparted to the apelin molecule,for example, the ability to link a functional moiety (e.g., PEG). Whenused in connection with amino acids, the symbol “U” shall mean“non-natural amino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” requirea modified tRNA and a modified tRNA synthetase (RS) for incorporationinto a protein. These “selected” orthogonal tRNA/RS pairs are generatedby a selection process as developed by Schultz et al. or by random ortargeted mutation. As way of example, pyrroline-carboxy-lysine is a“natural amino acid” as it is generated biosynthetically by genestransferred from one organism into the host cells and as it isincorporated into proteins by using natural tRNA and tRNA synthetasegenes, while p-aminophenylalanine (See, Generation of a bacterium with a21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L,Martin A B, King D S, Horn D M, Schultz P G. J Am Chem. Soc. 2003 Jan.29; 125(4):935-9) is an “unnatural amino acid” because, althoughgenerated biosynthetically, it is incorporated into proteins by a“selected” orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to,hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term “amino acid” also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, by way of example only, an α-carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group. Amino acid analoguesinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or their C-terminal carboxy group,their N-terminal amino group and/or their side-chain functional groupsare chemically modified. Such analogues include, but are not limited to,methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteinesulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alaninecarboxamide, homoserine, norleucine, and methionine methyl sulfonium.

TABLE 1 Un-natural or non-natural amino acids as described in theinvention: Symbol Name Structure Aib α-Aminoisobutyric acid

Nva or nva (D-Nva) L-Norvaline or D-Norvaline

1-Nal 1-Naphthalanine

2-Nal 2-Naphthalanine

Cha β-Cyclohexylalanine

Tic or tic (D-Tic) 1,2,3,4-Tetra- hydroisoquinoline- 3-carboxylic acid(D or L)

Nle or nle (D-Nle) L-Norleucine or D-Norleucine

pE Pyroglutamic acid

4-PhP 4-Phenylproline

Pip Pipecolinic acid

Abu or abu (D-Abu) 2-amino-butyric acid

3,4-Cl2F (D or L) 3,4- dichlorophenylalanine

5-Aminovaleric acid

O2Oc 8-Amino-3,6- dioxaoctanoic acid

Nal refers to both 1-Naphthalanine and 2-Naphthalanine, preferably2-naphthalanine. 4-Phenylproline refers to both cis and trans4-phenylproline, preferably trans-4-phenylproline

As used herein the term “amide” refers to an amide derivative of thecarboxylic acid group at the C-terminus (e.g. —C(O)NH₂, —C(O)NH—C₁₋₆alkyl, —C(O)NH—C₁₋₂alkylphenyl, —C(O)NH—NHBn, —C(O)-4 phenoxypiperidineor —C(O)N(C₁₋₆alkyl)₂).

The term “amide” also refers to derivatives of the amino group at theN-terminus (e.g. —NHC(O)C₁₋₁₆alkyl, —NHC(O)(CH₂)_(n)Ph (n is an integerof 1 to 6), —NHC(O)(CH₂)₂CO₂H, 4-Cl-Ph-(CH₂)₃C(O)NH—,C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—,C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—;C₁₆H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)NH—, Ph-CH₂CH₂NHC(O)—NH— orCH₃(OCH₂CH₂)_(m)C(O)NH— (m is an integer of 1 to 12).

As used herein, the term “ester” refers to an ester derivative of thecarboxylic acid group at the C-terminus (e.g —COOR) form wherein R ofthe ester refers to C₁₋₆ alkyl groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, etc., C₃₋₈ cycloalkyl groups such as cyclopentyl,cyclohexyl, etc., C₆₋₁₀ aryl groups such as phenyl, α-naphthyl, etc.,C₈₋₁₀ aryl-C₁₋₆ alkyl groups, for example phenyl-C₁₋₂ alkyl groups suchas benzyl, phenethyl, benzhydryl, etc., and α-naphthyl-C₁₋₂ alkyl groupssuch as α-naphthylmethyl and the like. Mention may also be made ofpivaloyloxymethyl ester and the like, which are commonly used as estersfor oral administration. When the polypeptides of the invention possessadditional carboxyl or carboxylate groups in positions other than the Cterminus, those polypeptides in which such groups are amidated oresterified also fall under the category of the polypeptide of theinvention. In such cases, the esters may for example be the same kindsof esters as the C-terminal esters mentioned above.

The term “APJ” (also referred to as “apelin receptor,”“angiotension-like-1 receptor,” “angiotension II-like-1 receptor,” andthe like) indicates a 380 residue, 7 transmembrane domain, Gi coupledreceptor whose gene is localized on the long arm of chromosome 11 inhumans (NCBI Reference Sequence: NP_(—)005152.1, and encoded by NCBIReference Sequence: NM_(—)005161). APJ was first cloned in 1993 fromgenomic human DNA using degenerate oligonucleotide primers (O'Dowd etal. Gene, 136:355-60, 1993) and shares significant homology withangiotensin II receptor type 1. Despite this homology however,angiotensin II does not bind APJ. Although orphan for many years, theendogenous ligand has been isolated and named apelin (Tatemoto et al.,Biochem Biophys Res Commun 251, 471-6 (1998)).

The term “apelin,” indicates a 77 residue preprotein (NCBI ReferenceSequence: NP_(—)0059109.3, and encoded by NCBI Reference Sequence:NM_(—)017413.3), which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12. The full length mature peptide, referred to as “apelin-36,”comprises 36 amino acids, but the most potent isoform is thepyroglutamated form of a 13mer of apelin (apelin-13), referred to as“Pyr-1-apelin-13 or Pyr¹-apelin-13” Different apelin forms aredescribed, for instance, in U.S. Pat. No. 6,492,324B1.

Polypeptides of the Invention:

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments.

In embodiment 1, the invention therefore provides a peptide or apolypeptide formula (I) (SEQ ID NO: 1):X1-X2-X3-R—X5-X6-X7-X8-X9-X10-X11-X12-X13  Iwherein:X1 is the N-terminus of the polypeptide and is either absent or pE;X2 is R or r;X3 is P or 4-PhP;X5 is L, Cha, D-L, F, Y, Y(Bzl), 3,4-Cl2-F or Nal;X6 is a D-amino acid, S or A;X7 is a D-amino acid, L, H or Aib; and at least one of X6 and X7 isD-amino acid or Aib;X8 is K, k, Q or E;X9 is G or D;X10 is P or pipecolic acid;X11 is D-Nle, Nle, f or D-Nva;X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 andX13 is a D-amino acid;wherein:Nle is L-norleucine;D-Nle is D-norleucine;Nal is L-(naphthyl)alanine;D-Nva is D-norvaline;Aib is α-aminoisobutyric acid;Cha is (S)-β-cyclohexylalanine;D-Tic is D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;pE is L-pyroglutamic acid;3,4-Cl2-F is (S)-3,4-dichlorophenylalanine;Y is L-tyrosine; andY(Bzl) is L-benzyl-tyrosine;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 1a, the invention therefore provides a peptide or apolypeptide formula (IA) (SEQ ID NO: 2):X1-X2-X3-R—X5-X6-X7-X8-G-X10-X11-X12-X13  IAwherein:X1 is the N-terminus of the polypeptide and is either absent or pE;X2 is R or r;X3 is P or 4-PhP;X5 is L, Cha, D-L, F, Y, Y(Bzl), 3,4-Cl2-F or 2-Nal;X6 is a D-amino acid or S;X7 is a D-amino acid, H or Aib; and at least one of X6 and X7 is D-aminoacid or Aib;X8 is K or k;X10 is P or pipecolic acid;X11 is D-Nle or Nle;X12 is absent, P or a D-amino acid;X13 is the C-terminus and is absent, F or a D-amino acid; and at leastone of X11, X12 andX13 is a D-amino acid;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In one aspect of embodiment 1 or 1A, the invention pertains to peptideor polypeptide of Formula I or IA wherein the amino group in the sidechain of K or k is optionally linked to a fatty acid via an amide bond.In a further aspect of this embodiment, the fatty acid is selected fromlauroyl, myristoyl or palmitoyl, wherein lauroyl is C₁₁H₂₃C(O)—,myristoyl is C₁₃H₂₇C(O)— and palmitoyl is C₁₅H₃₁C(O)—.

In one aspect of embodiment 1 or 1A, the invention pertains to peptideor polypeptide of Formula I or IA wherein the amino group in the sidechain of K or k is optionally linked to a lipophilic group via an amidebond, wherein the lipophilic group is selected from a fatty acid asdescribed supra and lauroyl(O2Oc), myristoyl(O2Oc) and palmitoyl(O2Oc)and wherein lauroyl(O2Oc) is C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—;myristoyl(O2Oc) is C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—;palmitoyl(O2Oc) is C₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—. Examples ofside chain fatty acids have been described in U.S. provisionalapplication No. 61/591,557 filed on Jan. 27, 2012, which is herebyincorporated by reference.

In embodiment 2, the invention pertains to a polypeptide according toembodiment 1 or 1A wherein X6 and X12 are D-amino acids (SEQ ID NO: 3);or an amide, an ester or a salt thereof.

In embodiment 3, the invention pertains to a polypeptide according toembodiment 2, wherein X13 is D-amino acid (SEQ ID NO: 4); or an amide,an ester or a salt thereof.

In embodiment 4, the invention pertains to a polypeptide according toembodiment 3, wherein X11 is D-amino acid (SEQ ID NO: 5); or an amide,an ester of a salt thereof.

In embodiment 5, the invention pertains to a polypeptide according toembodiment 1 or 1A, wherein X6 and X13 are D-amino acids (SEQ ID NO: 6);or an amide, an ester or a salt thereof.

In embodiment 6, the invention pertains to a polypeptide according toembodiment 1, wherein X7 and X12 are D-amino acids (SEQ ID NO: 7); or anamide, an ester or a salt thereof.

In embodiment 7, the invention pertains to a polypeptide according toembodiment 6 wherein X13 is a D-amino acid (SEQ ID NO: 8); or an amide,an ester or a salt thereof.

In embodiment 8, the invention pertains to a polypeptide accordinganyone of to embodiments 1 to 7 having the following formula II (SEQ IDNO: 9):X1-R—P—R—X5-a-X7-X8-G-P—X11-X12-X13  IIor an amide, an ester or a salt of the polypeptide.

In embodiment 9, the invention pertains to a polypeptide according toanyone of embodiments 1 to 8 having Formula III (SEQ ID NO: 10):X1-R—P—R—X5-X6-X7-K-G-P—X11-a-f  III;or an amide, an ester or a salt of the polypeptide.

In embodiment 10, the invention pertains to a polypeptide according toanyone of embodiments 1, 6, 7 and 9 having Formula IV (SEQ ID NO: 11):X1-R—P—R—X5-S—X7-K-G-P—X11-X12-X13  IV;or an amide, an ester or a salt of the polypeptide.

In embodiment 11, the invention pertains to a polypeptide according toanyone of embodiments 1 to 9, wherein X6 is a D-amino acid selected froma, D-Leu, k, s, d, nva, abu, f, h, v and D-Cys(tBu) (SEQ ID NO: 12); oran amide, an ester or a salt thereof. In a further aspect of thisembodiment, X11 is nle or f.

In embodiment 12, the invention pertains to a polypeptide according toanyone of embodiments 1 to 11 wherein X7 is Aib or a D-amino acidselected from a, f and (SEQ ID NO: 13); or an amide, an ester or a saltthereof.

In embodiment 13, the invention pertains to a polypeptide according toanyone of embodiments 1 to 12 wherein X12 is absent or a D-amino acidselected from a, f, p, e, r, abu, nva, and D-Leu (SEQ ID NO: 14); or anamide, an ester or a salt thereof. In a further aspect of thisembodiment, X12 is a.

In embodiment 14, the invention pertains to a polypeptide according toanyone of embodiments 1 to 13 wherein X13 is absent or is a D-amino acidselected from f, y, d, and D-Tic (SEQ ID NO: 15), or an amide, an esteror a salt thereof. In a further aspect of this embodiment X13 is f.

In embodiment 15, the invention pertains to a polypeptide according toembodiment 14 wherein X13 is absent or f (SEQ ID NO: 16); or an amide,an ester or a salt of the polypeptide.

In embodiment 16, the invention pertains to a polypeptide according toanyone of the preceding embodiments wherein X1 is pE (SEQ ID NO: 17); oran amide, an ester or a salt of the polypeptide.

In embodiment 17, the invention pertains to a polypeptide according toanyone of embodiments 1 to 16 wherein X5 is L (SEQ ID NO: 18); or anamide, an ester or a salt of the polypeptide.

In embodiment 18, the invention pertains to a polypeptide according toanyone of embodiments 1 to 17 wherein X8 is K (SEQ ID NO: 19); or anamide, an ester or a salt of the polypeptide.

In embodiment 19, the invention pertains to a polypeptide according toanyone of embodiments 1 to 18 wherein X11 is Nle or nle (SEQ ID NO: 20);or an amide, an ester or a salt of the polypeptide.

In embodiment 20, the invention pertains to a polypeptide according toanyone of the preceding embodiments wherein the C-terminus is an amide;or a salt of the polypeptide.

In embodiment 21, the invention pertains to a polypeptide according toembodiment 20 wherein the C-terminus is an amide of formula —C(O)—R2 andR2 is —NH₂, —NH—(CH₂)₂-Ph or 4-phenoxypiperidine; or a salt of thepolypeptide.

In another embodiment, the invention pertains to peptides andpolypeptides according to any one of Formulae I, IA, II, III or IV, orany of any other classes and subclasses described supra, (i.e. accordingto anyone of the embodiments 1 to 15 and 17-21) or an amide, an ester ora salt thereof, wherein X1 is absent; or an amide, an ester or a salt ofthe polypeptide. In one aspect of this embodiment, the N-terminus of thepeptide is an amide. In a further aspect of this embodiment, theinvention pertains to peptides and polypeptide according to any one ofFormulae I, IA, II, III or IV, or any of any other classes andsubclasses described supra, or an amide, an ester or a salt thereof,wherein the X1 is absent and the N-terminus is an amide of formula —NHRand R is CH₃C(O)—, CH₃—(O—CH₂CH₂)_(m)—C(O)—, palmitoyl(O2Oc)_(p),myristoyl(O2Oc)_(p), lauroyl(O2Oc)_(p) or Ph-CH₂CH₂NHC(O)—, benzoyl,phenacyl, succinyl, octanoyl, 4-phenylbutanoyl, 4-Cl-Ph-(CH₂)₃C(O)—, orPh-CH₂CH₂NHC(O)—; and wherein

p is an interger 1 to 4;

m is an integer 1 to 12;

Lauroyl(O2Oc)_(p) is C₁₁H₂₃C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;

Myristoyl(O2Oc)_(p) is C₁₃H₂₇C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;

Palmitoyl(O2Oc)_(p) is C₁₅H₃₁C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—.In one particular aspect of this embodiment R is acetyl, benzoyl,phenacyl, succinyl, octanoyl, 4-phenylbutanoyl, 4-Cl-Ph-(CH₂)₃C(O)—, orPh-CH₂CH₂NHC(O)—. Examples of N-terminus amides have been described inU.S. provisional application No. 61/591,557 filed on Jan. 27, 2012,which is hereby incorporated by reference.

In another embodiment, the invention pertains to peptides orpolypeptides according to anyone of Formulae I, IA, II, III or IV, or toany other classes and subclasses described supra, (i.e. according toanyone of embodiments 1 to 21), or an amide, an ester or a salt thereof,wherein N-terminus is an amide of formula NHR1 wherein R1 is CH₃C(O)—,CH₃—(O—CH₂CH₂)_(m)—C(O)—, palmitoyl(O2Oc), myristoyl(O2Oc),lauroyl(O2Oc) or Ph-CH₂CH₂NHC(O)—; and wherein m, lauroyl(O2Oc),myristoyl(O2Oc) and palmitoyl(O2Oc) are defined supra.

In another embodiment, the invention pertains to peptides andpolypeptides according to any one of Formulae I, IA, II, III or IV, orany of any other classes and subclasses described supra, (i.e. accordingto anyone of embodiments 1 to 8 and 10 to 21), wherein X13 is absent; oran amide, an ester or a salt thereof. In a particular aspect of thisembodiment, the C-terminus is an amide. In a further aspect of thisembodiment, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I, IA, II, III or IV, or any of anyother classes and subclasses described supra, or an amide, an ester or asalt thereof, wherein the C-terminus is an amide of formula —C(O)R2 andR2 is —NH₂, —NH-Me, —NH—NHBn, 4-Phenoxypiperidin-1-yl or —NH—(CH₂)₂-Ph.In a preferred aspect of this embodiment, the invention pertains topeptides and polypeptide according to any one of Formulae I to IV, orany of any other classes and subclasses described supra, or an amide, anester or a salt thereof, wherein the C-terminus is an amide of formula—C(O)R2 and R2 is —NH₂, —NH—(CH₂)₂-Ph or 4-phenoxypiperidin-1-yl.

In one embodiment, the invention pertains to a peptide or polypeptide ofanyone of embodiments 1 to 21, wherein three of the amino acids X1 toX13 are different from the corresponding amino acids present inPyr-1-apelin-13. In another embodiment, the invention pertains to apeptide or polypeptide of anyone of embodiments 1 to 21 wherein four ofthe amino acids X1 to X13 are different from the corresponding aminoacids present in Pyr-1-apelin-13.

In another embodiment, X1, X2, X3, X5, X6, X7, X8. X9, X10, X11, X12 andX13 amino acids are those defined by X1, X2, X3, X5, X6, X7, X8. X9,X10, X11, X12 and X13 amino acids in the Examples section below.

In another embodiment, individual polypeptides according to theinvention are those listed in the Examples section below or apharmaceutically acceptable salt thereof.

Unless specified otherwise, the term “polypeptide of the presentinvention” refers to a polypeptide of Formula (I) and subformulaethereof (Formulae IA, II, III or IV); or an amide, an ester or a saltthereof.

Unless specified otherwise, the terms “polypeptides of the presentinvention,” “peptides of the present invention,” “apelin peptideagonists,” and the like refer to peptides and polypeptides of Formula Iand subformulae thereof (Formulae IA, II, III or IV); or an amide, anester or a salt thereof. The peptides and polypeptides of the inventiondemonstrate substantially equivalent or improved activity and/or plasmastability over known apelin peptides and polypeptides described herein,including but not limited to wild type apelin, apelin-13 andpyr-1-apelin-13.

The peptides and polypeptides of the invention also encompass peptidesand polypeptides that are at least about 95% identical to the peptidesand polypeptides according to any one of Formulae I, IA, II, III or IV,or an amide, an ester or a salt thereof, as well as to any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

As used herein, the phrase “homologous amino acid sequence,” orvariations thereof, refers to sequences characterized by a homology, atthe amino acid level, of at least a specified percentage and is usedinterchangeably with “sequence identity.” Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, an amino acid sequence ishomologous if it has at least 60% or greater, up to 99%, identity with acomparator sequence. In some embodiments, an amino acid sequence ishomologous if it shares one or more, up to 60, amino acid substitutions,additions, or deletions with a comparator sequence. In some embodiments,the homologous amino acid sequences have no more than 5 or no more than3 conservative amino acid substitutions.

Homology may also be at the polypeptide level. The degree or percentageidentity of peptides or polypeptides of the invention, or portionsthereof, and different amino acid sequences is calculated as the numberof exact matches in an alignment of the two sequences divided by thelength of the “invention sequence” or the “foreign sequence”, whicheveris shortest. The result is expressed as percent identity.

A polypeptide comprising an amino acid sequence having a homology ofabout 80-99.9%, preferably 90-99.9% to the amino acid sequence describedin the specific examples, and possessing a plasma stability superior toapelin-13 or pyr-1-apelin-13, fall under the category of the polypeptideof the invention. In one embodiment, the plasma stability improvement isat least 2 fold. In one embodiment, the polypeptide of the invention hasa plasma stability of at least 30 minutes. In another embodiment, thepolypeptide of the invention has a plasma stability of at least 60minutes, or at least 80 minutes, preferably at least 100 min and morepreferably at least 150 minutes.

The term “substantially equivalent” means the nature of thereceptor-binding activity, signal transduction activity and the like isequivalent. Thus, it is allowable that even differences among gradessuch as the strength of receptor binding activity and the molecularweight of the polypeptide are present.

A polypeptide as described herein, or a substantial equivalent thereto,by substitution, deletion, addition or insertion of one or more of aminoacids may be mentioned as polypeptides containing an amino acid sequencesubstantial equivalent(s) in the above sense. A polypeptide as describedherein, or a substantial equivalent thereto, by substitution of 1 to 5,preferably 1 to 3 and more preferably 1 or 2 amino acids with natural orun-natural amino acids may be mentioned as polypeptides containing anamino acid sequence substantial equivalent(s) in the above sense.Further modifications and alterations may include the replacement of anL-amino-acid with a D-amino acid, or other variation including, but notlimited to, phosphorylation, carboxylation, alkylation and the like aslong as the apelin agonistic activity of the peptide or polypeptide offormulae I, IA, II, III or IV is maintained and the plasma stability isimproved over the pyroglutamated form of apelin-13. For example, D-aminoacid are well tolerated with respect to activity and stability of thepolypeptide at position 2 (X2), position 6 (X6), position 7 (X7),position 8 (X8), position 11 (X11), position 12 (X12) and position 13(X13) of the linear peptides and polypeptides of Formulae I, IA, II, IIIor IV.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thepolypeptides of this invention and, which typically are not biologicallyor otherwise undesirable. In many cases, the polypeptides of the presentinvention are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulformate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

The term “a therapeutically effective amount” of a polypeptide of thepresent invention refers to an amount of the polypeptide of the presentinvention that will elicit the biological or medical response of asubject, for example, amelioration of a symptom, alleviation of acondition, slow or delay disease progression, or prevention of adisease, etc. In one non-limiting embodiment, the term “atherapeutically effective amount” refers to the amount of thepolypeptide of the present invention that, when administered to asubject, is effective to (1) at least partially alleviate, inhibit,prevent and/or ameliorate a condition, a disorder or a disease or asymptom thereof (i) ameliorated by the activation of the APJ receptor or(ii) associated with the activity of the APJ receptor, or (iii)characterized by abnormal activity of the APJ receptor; or (2) activatethe APJ receptor.

In another non-limiting embodiment, the term “a therapeuticallyeffective amount” refers to the amount of the polypeptide of the presentinvention that, when administered to a cell, or a tissue, or anon-cellular biological material, or a medium, is effective to at leastpartially activate the APJ receptor. As will be appreciated by those ofordinary skill in the art, the absolute amount of a particular agentthat is effective may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the target tissue, etc.Those of ordinary skill in the art understand that “a therapeuticallyeffective amount” may be administered in a single dose or may beachieved by administration of multiple doses. For example, in the caseof an agent to treat heartfailure, an effective amount may be an amountsufficient to result in clinical improvement of the patient, e.g.,increased exercise tolerance/capacity, increased blood pressure,decrease fluid retention, and/or improved results on a quantitative testof cardiac functioning, e.g., ejection fraction, exercise capacity (timeto exhaustion), etc.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In certain embodiments, the subject is aprimate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence, onset, or development of one ormore symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a therapeutic agent), or theadministration of a combination of therapies (e.g., a combination oftherapeutic agents).

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The peptides and polypeptides of the present invention can be producedby the per se known procedures for peptide synthesis. The methods forpeptide synthesis may be any of a solid-phase synthesis and aliquid-phase synthesis. Thus, the peptide and polypeptide of interestcan be produced by condensing a partial peptide or amino acid capable ofconstituting the protein with the residual part thereof and, when theproduct has a protective group, the protective group is detachedwhereupon a desired peptide can be manufactured. The known methods forcondensation and deprotection include the procedures described in thefollowing literature (1)-(5).

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966,-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,    1965,-   (3) Nobuo Izumiya et al. Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975,-   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Protein Chemistry IV, 205, 1977, and-   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,    Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide can be purified and isolated by acombination of conventional purification techniques such as solventextraction, column chromatography, liquid chromatography, andrecrystallization. Where the peptide isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenz-hydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 2-chlorotritylchloride resin, and so on. Using such a resin, amino acids whose α-aminogroups and functional groups of side-chain have been suitably protectedare condensed on the resin according to the sequence of the objectivepeptide by various condensation techniques which are known per se. Atthe end of the series of reactions, the peptide or the protected peptideis removed from the resin and the protective groups are removed and ifnecessary, disulfide bonds are formed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used such asHATU, HCTU or e.g. a carbodiimide. The carbodiimide includes DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt or Oxyma Purecan be used. The protected amino acid can be directly added to the resinalong with the activation reagents and racemization inhibitor or bepre-activated as symmetric acid anhydride, HOBt ester, or HOOBt esterthen added to the resin. The solvent for the activation of protectedamino acids or condensation with the resin can be properly selected fromamong those solvents which are known to be useful for peptidecondensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned.

The reaction temperature can be selected from the range hitherto-knownto be useful for peptide bond formation and is usually selected from therange of about −20° C.-50° C. The activated amino acid derivative isgenerally used in a proportion of 1.5-4 fold excess. If the condensationis found to be insufficient by a test utilizing the ninhydrin reaction,the condensation reaction can be repeated to achieve a sufficientcondensation without removing the protective group. If repeatedcondensation still fails to provide a sufficient degree of condensation,the unreacted amino group can be acetylated with acetic anhydride oracetyllmidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiary-amyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₆₋₁₀aryl-C₇₋₂alkyl as well as 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, andtritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-tri ethylbenzenesulfonyl, DNP, benzyloxymethyl, Burn,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide. To obtain an ester of the polypeptide, the α-carboxyl groupof the C-terminal amino acid is condensed with a desired alcohol to givean amino acid ester and then, the procedure described above forproduction of the amide is followed.

The polypeptides of the instant invention, or an amide, an ester of asalt thereof, may be administered in any of a variety of ways, includingsubcutaneously, intramuscularly, intravenously, intraperitoneally,inhalationally, etc. Particularly preferred embodiments of the inventionemploy continuous intravenous administration of the polypeptides of theinstant invention, or an amide, ester, or salt thereof. The polypeptideson the instant invention may be administered as a bolus or as acontinuous infusion over a period of time. An implantable pump may beused. In certain embodiments of the invention, intermittent orcontinuous polypeptides administration is continued for one to severaldays (e.g., 2-3 or more days), or for longer periods of time, e.g.,weeks, months, or years. In some embodiments, intermittent or continuouspolypeptide administration is provided for at least about 3 days. Inother embodiments, intermittent or continuous polypeptide administrationis provided for at least about one week. In other embodiments,intermittent or continuous polypeptide administration is provided for atleast about two weeks. It may be desirable to maintain an average plasmapolypeptide concentration above a particular threshold value eitherduring administration or between administration of multiple doses. Adesirable concentration may be determined, for example, based on thesubject's physiological condition, disease severity, etc. Such desirablevalue(s) can be identified by performing standard clinical trials.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a polypeptide of the present invention or andamide, an ester or a salt thereof and one or more pharmaceuticallyacceptable carriers. The pharmaceutical composition can be formulatedfor particular routes of administration such as oral administration,parenteral administration, and rectal administration, etc. In addition,the pharmaceutical compositions of the present invention can be made upin a solid form (including without limitation capsules, tablets, pills,granules, powders or suppositories), or in a liquid form (includingwithout limitation solutions, suspensions or emulsions). Thepharmaceutical compositions can be subjected to conventionalpharmaceutical operations such as sterilization and/or can containconventional inert diluents, lubricating agents, or buffering agents, aswell as adjuvants, such as preservatives, stabilizers, wetting agents,emulsifers and buffers, etc.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Preferred pharmaceutical formulations are stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive therapeutic agents arepreferably delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. It is noted that the lungsprovide a large surface area for systemic delivery of therapeuticagents.

The agents may be encapsulated, e.g., in polymeric microparticles suchas those described in U.S. publication 20040096403, or in associationwith any of a wide variety of other drug delivery vehicles that areknown in the art. In other embodiments of the invention the agents aredelivered in association with a charged lipid as described, for example,in U.S. publication 20040062718. It is noted that the latter system hasbeen used for administration of a therapeutic polypeptide, insulin,demonstrating the utility of this system for administration of peptideagents.

Systemic administration can also be by transmucosal or transdermalmeans.

Suitable compositions for transdermal application include an effectiveamount of a polypeptide of the invention with a suitable carrier.Carriers suitable for transdermal delivery include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound of the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g., for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication. They are thus particularly suited for use in topical,including cosmetic, formulations well-known in the art. Such may containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

Method of the Invention:

Apelin family of peptides is the only known natural family of ligandsfor the G protein coupled APJ receptor. Apelin gene encodes a 77aminoacid polypeptide, which gets processed into biologically activeforms of apelin peptides, such as apelin-36, apelin-17, apelin-16,apelin-13, apelin-12 and pyroglutamate modified form of apelin-13(Pyr¹-apelin-13). Any one of these apelin peptides, upon binding to APJreceptor, transduces the signal via Gi and Gq proteins. Incardiomyocytes, Gi or Gq coupling leads to changes in intracellular pH,PLC activation, and IP3 production that enhance myofilament calciumsensitivity and ultimately result in increased cardiac contractility. Gicoupling inhibits activated Gs, adenylyl cyclase and cAMP production andincreases pAkt levels leading to cardioprotection. In vascularendothelial cells, APJ activation via Gi, pAKT leads to increased nitricoxide (NO) production, which increases smooth muscle relaxationresulting in overall vasodilation.

Patients with chronic stable heart failure have occasional acuteepisodes of decompensation, where cardiac contractility declines furtherand symptoms worsen. These exacerbations are referred to as acutedecompensated heart failure (ADHF). Current therapies for ADHF includediuretics, vasodilators, and inotropes, which directly increase cardiaccontractility. Current intravenous inotropes (dobutamine, dopamine,milrinone, levosimendan) are well known for their adverse events such asarrhythmia and increased long-term mortality. The synthetic apelinpolypeptide analogs of the instant invention provide a therapy for ADHFthat increases cardiac contractility without arrhythmogenic or mortalityliabilities and address the enormous unmet medical need in chronic heartfailure.

Indeed, acute apelin treatment (5 min) in humans results in coronaryvasodilatation and improved cardiac output. However, native apelinsexhibit a very short t_(1/2) (seconds) and duration of action (fewminutes) in vivo. The potent synthetic apelin peptide agonists of theinstant invention have longer half lives compared to the native apelin.

Activation of APJ receptor in cardiomyocytes a) improve cardiaccontractility via Gi/Gq, PLC and Ca2+, and b) provide cardioprotectionvia Gi, pAkt activation, but without increasing cAMP (as seen with otherinotropes). In addition, APJ agonism in endothelial cells leads toarterial vasodilation, which further benefits heart failure by unloadingthe work of left ventricle. Taken together the synthetic apelinpolypeptide analogs can improve overall cardiac function, reducearrhythmogenesis and provide survival benefit.

More recently, there have been a number of preclinical researchpublications focusing on the potential involvement of Apelin in diabetesand insulin resistance. Apelin has been shown to 1) lower blood glucoselevels by improving glucose uptake in muscle, adipose and heart, 2)protect pancreatic beta cells from ER stress and subsequent apoptosis,3) lower the insulin secretion in beta cells, and 4) regulatecatecholamine induced lypolysis in adipose tissue. Activation of pAKTpathway has been implicated in these processes.

The polypeptides according to anyone of formulae I to IV, or apharmaceutically acceptable salt thereof, in free form or inpharmaceutically acceptable salt form, exhibit valuable pharmacologicalproperties, e.g. APJ receptor agonsim properties, e.g. as indicated inin vitro and in vivo tests as provided in the next sections and aretherefore indicated for therapy.

Polypeptides of the invention or a pharmaceutically acceptable saltthereof, may be useful in the treatment of an indication selected fromacute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof for the treatment of a disease which is associated with theAPJ receptor activity. In a further embodiment, the therapy is selectedfrom a disease which is responsive to the agonism of the APJ receptor.In another embodiment, the disease is selected from the afore-mentionedlist, suitably acute decompensated heart failure. In yet another subsetof this embodiment, the present invention provides the use of apolypeptide of anyone of formulae I to IV, or an amide, ester or a saltthereof, in the manufacture of a medicament, for the treatment of adisease which is associated with the APJ receptor activity.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IV, or an amide, an ester or asalt thereof, in therapy. In a further embodiment, the therapy isselected from a disease which may be treated by activation (agonism) ofthe APJ receptor.

In another embodiment, the invention provides a method of treating adisease which is responsive to the agonism of the APJ receptor,comprising administration of a therapeutically acceptable amount of apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof. In a further embodiment, the disease is selected from theafore-mentioned list, suitably acute decompensated heart failure.

In yet another subset of this embodiment, the invention provides amethod of treating a disease which is associated with the activity ofthe APJ receptor comprising administration of a therapeuticallyacceptable amount of a polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof.

The effective amount of a pharmaceutical composition or combination ofthe invention to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the fusion protein variant is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The activity of a polypeptide according to the present invention can beassessed by the following in vitro methods described below.

hAPJ Calcium Flux Assay:

Chem-5 APJ stable cells (Millipore # HTS068C) were plated in 384-wellformat with 10,000 cells/well in 25 ul growth media, then grown 24 hoursin a 37° C. tissue culture incubator. One hour before the assay, 25ul/well FLIPR Calcium 4 dye (Molecular Devices R8142) with 2.5 mMprobenecid was added, and cells were incubated one hour in a 37° C.tissue culture incubator. Peptides were solubilized in HBSS, HEPES &0.1% BSA buffer, and serially-diluted 10-fold, from 50 uM to 5 μM, intriplicate. FLIPR Tetra was used to add peptide to the cells with dye(1:5, for final peptide concentrations ranging from 10 uM to 1 μM).FLIPR dye inside the cells emitted fluorescence after binding tocalcium, while fluorescence from outside the cells was masked.Fluorescence was measured using 470-495 excitation and 515-575 emissionwavelengths on the FLIPR Tetra. Readings were done for 3 minutes total,beginning 10 seconds before the peptide addition. Maximum-minimum valueswere calculated and plotted for each peptide concentration, and GraphPadprism software was used to calculate EC₅₀ values at the curve inflectionpoints, for calcium flux stimulation by peptides.

Plasma Stability Assay:

Materials:

Working solution: 1 mg/mL test article is prepared in Milli-Q water

Extraction solution: Methanol:Acetonitrile:Water (1:1:1) with 0.1%Formic Acid and 400 ng/mL Glyburide.

Plasma: Male Sprague-Dawley rat plasma (with sodium heparin), purchasedfrom Bioreclamation LLC (Liverpool, N.Y.).

Whole blood: Male Sprague Dawley whole blood (with sodium heparin),purchased from Bioreclamation LLC (Liverpool, N.Y.)

Lung homogenate: Male rat Sprague Dawley lung was purchased fromBioreclamation LLC (Liverpool, N.Y.). The lung was homogenized usingpolytron homogenizer after addition of 5× volume of 1×PBS. Thehomogenate was centrifuged at 9000 rpm for 10 min at 4° C. Thesupernatant was centrifuged again at 3000 rpm for 30 min to make a clearsupernatant. Protein concentration was determined using a commercial kit(Pierce, Thermo Scientific).Sample Preparation Procedure:

Test article was prepared in one of the following biological matrices:heparinized rat plasma, heparinized rat whole blood or lung homogenate.The plasma and whole blood sample was prepared at 5000 ng/mL by adding 5uL of 1 mg/mL Working solution to 995 uL of rat plasma or whole blood.Lung homogenate samples were prepared by diluting lung homogenate to 1mg/ml protein concentration with phosphate buffered saline (PBS),followed by addition of 5 uL Working solution to 995 uL diluted lunghomogenate. The samples were incubated at 37° C. with gentle shaking(65˜75 rpm) in a water bath incubator. At times 0 min, 5 min, 15 min, 30min, 60 min, 120 and 240 min, 25 uL aliquots of incubation samples weretransferred to 96-well plate and immediately protein precipitated using150 uL of Extraction solution. After completion of incubationexperiment, the sample plate was centrifuged at 4000 rpm at 4° C. for 10minutes. Afterwards, a pipetting device (Tecan Temo) was used to tranferthe supernatants to another plate and add 50 uL of water to all samples.The plate was vortexed prior to LC-MS analysis.

LC-MS Analysis of stability samples

HPLC: Agilent 1290 HPLC with autosampler

Column: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.

Mobile phase A: 0.1% Formic acid in acetonitrile

Mobile phase B: 0.1% Formic acid in water

Gradient Program:

Time (min) Flow (mL) Mobile Phase A (%) Mobile Phase B (%) 0 0.4 95 50.5 0.4 95 5 1.5 0.4 5 95 4.1 0.4 5 95 4.2 0.4 95 5 5 0.4 95 5Mass spectrometer: Agilent Q-TOF 6530Data acquisition mode: Full scan with mass range of 100-1000 m/zData acquisition and analysis software: MassHunterData Analysis:Stability assay: stability half-life, (t½), values were determined byconverting peak areas at each time point to percent remaining relativeto initial (t=0) peak area.Percent remaining=100×(sample peak area)÷(t=0 peak area)The natural log of percent remaining values were calculated and plottedagainst sample time (Microsoft Excel). The slope of this line, k, wasdetermined by linear regression (Microsoft Excel).Stability half-life was then calculated by the formula, t½=0.693÷kSurrogate Activity-Based Plasma Stability Assay:

The calcium flux protocol described above was followed, with thefollowing changes. The peptides were also incubated with 5% rat plasma(Bioreclamation # RATPLNAHP-M, Na Heparin-treated). Readings were takenat time points t_(o) and t₂₄ hrs, after incubation in a 37° C. tissueculture incubator. Peptide plasma half-life in minutes was estimated bycalculating the following:

1) LN((EC₅₀ at t₀)/(EC₅₀ at t_(24 hrs))),

2) Calculate slope of value above and

3) t_(1/2)=0.693/(slope^2).

Using the test assay (as described above) polypeptides of the inventionexhibited efficacy and stability in accordance to Tables 2 and 3,provided infra.

TABLE 2 Activity and stability of polypeptides Surrogate hAPJ Ca²⁺activity-based Flux EC₅₀ Plasma stability Peptide [nM] t½ [min] Example1 2.3 >1000 Example 2 385.7 >1000 Example 3 796.5 >1000 Example 4 5.0730.4 Example 5 79.9 >1000 Example 6 3.1 >1000 Example 7 692.7 >1000Example 8 7.7 >1000 Example 9 9.9 >1000 Example 10 154.6 >1000 Example11 1063.8 >1000 Example 12 0.9 >1000 Example 13 1.1 >1000 Example 14 3.2184.7 Example 15 2.1 >1000 Example 16 8.8 409.0 Example 17 3.1 >1000Example 18 66.5 122.3 Example 19 169.5 >1000 Example 20 922.1 >1000Example 21 0.9 >1000 Example 22 86.8 >1000 Example 23 2.8 124.8 Example24 13.8 57.5 Example 25 69.2 241.5 Example 26 1.7 985.5 Example 27 120.9348.9 Example 28 113.8 >1000 Example 29 1.8 565 Example 30 2.4 128.5Example 31 99.0 346.6 Example 32 30.8 47.7 Example 33 14.8 >1000 Example34 34.5 23.2 Example 35 10.7 >1000 Example 36 16.2 121.4 Example 37 3.9523.5 Example 38 3.3 >1000 Example 39 4.5 888.5 Example 40 2.0 >1000Example 41 4.5 42.2 Example 42 2.4 >1000 Example 43 2.0 178 Example 443.0 555 Example 45 2.4 >1000 Example 46 9.2 532 Example 47 4.4 >1000Example 48 16.1 248 Example 49 623.0 >1000 Example 50 1.4 11 Example 518.5 >1000 Example 52 19.5 >1000 Example 53 8.7 >1000 Example 54 0.9 902Example 55 72.8 101 Example 56 6.8 331 Example 57 1.5 >1000 Example 5855.1 228 Example 59 7.4 849 Example 60 200.0 >1000 Example 61 7.9 >1000Example 62 95.9 474 Example 63 9.4 225 Comparative 1.8 5.0 Example:Pyr1-apelin-13

TABLE 3 Correlation between plasma stability assay and surrogateactivity based plasma stability assay: Plasma Surrogate Activitystability based Plasma Peptide t½ [min] stability t½ [min] Example 4 414730 Example 14 405 185 Example 16 448 409 Example 17 365 >1000 Example29 156 565 Example 30 121 128 Pyr-1-Apelin 13 6.6 5.0

The polypeptide of the present invention may have an APJ receptorpotency similar to apelin-13 or pyr-1-apelin-13. In one embodiment thepolypeptide of the present invention has an EC₅₀ of less than 100 nM. Inanother embodiment the polypeptide of the invention has an EC₅₀ of lessthan 50 nM, preferably less than 25 nM and more preferably less than 15nM. In yet another embodiment, the polypeptide of the present inventionhas an EC₅₀ of less than 10 nM.

The polypeptide of the present invention may have plasma stabilitysuperior to apelin-13 or pyr-1-apelin-13. In one embodiment, the plasmastability improvement is at least 2 fold. In one embodiment, thepolypeptide of the invention has a plasma stability of at least 30minutes. In another embodiment, the polypeptide of the invention has aplasma stability of at least 60 minutes, or at least 80 min, preferablyat least 100 minutes and more preferably at least 150 minutes.

The polypeptide of the present invention may be administered eithersimultaneously with, or before or after, one or more other therapeuticagent. The polypeptide of the present invention may be administeredseparately, by the same or different route of administration, ortogether in the same pharmaceutical composition as the other agents.

In one embodiment, the invention provides a product comprising apolypeptide of anyone of formulae I to IV, or an amide, an ester of asalt thereof, and at least one other therapeutic agent as a combinedpreparation for simultaneous, separate or sequential use in therapy. Inone embodiment, the therapy is the treatment of a disease or conditionresponsive to the activation of the APJ receptor.

Products provided as a combined preparation include a compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester of a salt thereof, and the other therapeutic agent(s) together inthe same pharmaceutical composition, or a polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof, and the othertherapeutic agent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a polypeptide of anyone of formulae I to IV, or an amide, anester or a salt thereof, and another therapeutic agent(s). Optionally,the pharmaceutical composition may comprise a pharmaceuticallyacceptable excipient, as described above.

In one embodiment, the invention provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains apolypeptide of anyone of formula I to IV, or an amide, an ester or asalt thereof. In one embodiment, the kit comprises means for separatelyretaining said compositions, such as a container, divided bottle, ordivided foil packet. An example of such a kit is a blister pack, astypically used for the packaging of tablets, capsules and the like.

The kit of the invention may be used for administering different dosageforms, for example, oral and parenteral, for administering the separatecompositions at different dosage intervals, or for titrating theseparate compositions against one another. To assist compliance, the kitof the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of theinvention and the other therapeutic agent may be manufactured and/orformulated by the same or different manufacturers. Moreover, thecompound of the invention and the other therapeutic may be broughttogether into a combination therapy: (i) prior to release of thecombination product to physicians (e.g. in the case of a kit comprisingthe compound of the invention and the other therapeutic agent); (ii) bythe physician themselves (or under the guidance of the physician)shortly before administration; (iii) in the patient themselves, e.g.during sequential administration of a polypeptide of the invention andthe other therapeutic agent.

Accordingly, the invention provides the use of a polypeptide of anyoneof formulae I to IV, or an amide, an ester or a salt thereof, fortreating a disease or condition responsive to the agonism of the APJreceptor, wherein the medicament is prepared for administration withanother therapeutic agent. The invention also provides the use ofanother therapeutic agent for treating a disease or condition responsiveto the agonism of the apelin receptor, wherein the medicament isadministered with a polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof.

The invention also provides a polypeptide of anyone of formulae I to IV,or a pharmaceutically acceptable salt thereof, for use in a method oftreating a disease or condition responsive to the agonism of the APJreceptor, wherein the polypeptide of anyone of formulae I to IV, or anamide, an ester or a salt thereof, is prepared for administration withanother therapeutic agent. The invention also provides anothertherapeutic agent for use in a method of treating a disease or conditionresponsive to the agonism of the APJ receptor, wherein the othertherapeutic agent is prepared for administration with a polypeptide ofanyone of formulae I to IV, or an amide, an ester or a salt thereof. Theinvention also provides a polypeptide of anyone of formulae I to IV, oran amide, an ester or a salt thereof, for use in a method of treating adisease or condition responsive to the agonism of the APJ receptor,wherein the polypeptide of anyone of formulae I to IV, or an amide, anester or a salt thereof, is administered with another therapeutic agent.The invention also provides another therapeutic agent for use in amethod of treating a disease or condition responsive to the agonism ofthe APJ receptor, wherein the other therapeutic agent is administeredwith a polypeptide of anyone of formulae I to IV or an amide, an esteror a salt thereof.

The invention also provides the use of a polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof, for treatinga disease or condition responsive to the agonism of the APJ receptor,wherein the patient has previously (e.g. within 24 hours) been treatedwith another therapeutic agent. The invention also provides the use ofanother therapeutic agent for treating a disease or condition responsiveto the agonism of the APJ receptor, wherein the patient has previously(e.g. within 24 hours) been treated with a polypeptide of anyone offormulae I to IV, or an amide, an ester or a salt thereof.

In one embodiment, the other therapeutic agent is selected frominotropes, beta adrenergic receptor blockers, HMG-Co-A reductaseinhibitors, angiotensin II receptor antagonists, angiotensin convertingenzyme (ACE) Inhibitors, calcium channel blockers (CCB), endothelinantagonists, renin inhibitors, diuretics, ApoA-I mimics, anti-diabeticagents, obesity-reducing agents, aldosterone receptor blockers,endothelin receptor blockers, aldosterone synthase inhibitors (ASI), aCETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide) and a NEPinhibitor.

The term “in combination with” a second agent or treatment includesco-administration of the polypeptide of the invention (e.g., apolypeptide according to anyone of Formulae I-IV or a polypeptideotherwise described herein) with the second agent or treatment,administration of the compound of the invention first, followed by thesecond agent or treatment and administration of the second agent ortreatment first, followed by the compound of the invention.

The term “second agent” includes any agent which is known in the art totreat, prevent, or reduce the symptoms of a disease or disorderdescribed herein, e.g. a disorder or disease responsive to the actvationof the APJ receptor, such as for example, acute decompensated heartfailure (ADHF), chronic heart failure, pulmonary hypertension, atrialfibrillation, Brugada syndrome, ventricular tachycardia,atherosclerosis, hypertension, restenosis, ischemic cardiovasculardiseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention,diabetes (including gestational diabetes), obesity, peripheral arterialdisease, cerebrovascular accidents, transient ischemic attacks,traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries(including sunburn) and preeclampsia.

Examples of second agents include inotropes, beta adrenergic receptorblockers, HMG-Co-A reductase inhibitors, angiotensin II receptorantagonists, angiotensin converting enzyme (ACE) Inhibitors, calciumchannel blockers (CCB), endothelin antagonists, renin inhibitors,diuretics, ApoA-I mimics, anti-diabetic agents, obesity-reducing agents,aldosterone receptor blockers, endothelin receptor blockers, aldosteronesynthase inhibitors (ASI), a CETP inhibitor, anti-coagulants, relaxin,BNP (nesiritide) and/or a NEP inhibitor.

Inotropes as used herein include for example dobutamine, isoproterenol,milrinone, amirinone, levosimendan, epinephrine, norepinephrine,isoproterenol and digoxin.

Beta adrenergic receptor blockers as used herein include for exampleacebutolol, atenolol, betaxolol, bisoprolol, carteolol, metoprolol,nadolol, propranolol, sotalol and timolol.

Anti-coagulants as used herein include Dalteparin, Danaparoid,Enoxaparin, Heparin, Tinzaparin, Warfarin.

The term “HMG-Co-A reductase inhibitor” (also calledbeta-hydroxy-beta-methylglutaryl-co-enzyme-A reductase inhibitors)includes active agents that may be used to lower the lipid levelsincluding cholesterol in blood. Examples include atorvastatin,cerivastatin, compactin, dalvastatin, dihydrocompactin, fluindostatin,fluvastatin, lovastatin, pitavastatin, mevastatin, pravastatin,rosuvastatin, rivastatin, simvastatin, and velostatin, or,pharmaceutically acceptable salts thereof.

The term “ACE-inhibitor” (also called angiotensin converting enzymeinhibitors) includes molecules that interrupt the enzymatic degradationof angiotensin I to angiotensin II. Such compounds may be used for theregulation of blood pressure and for the treatment of congestive heartfailure. Examples include alacepril, benazepril, benazeprilat,captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat,fosinopril, imidapril, lisinopril, moexipril, moveltopril, perindopril,quinapril, ramipril, spirapril, temocapril, and trandolapril, or,pharmaceutically acceptables salt thereof.

The term “endothelin antagonist” includes bosentan (cf. EP 526708 A),tezosentan (cf. WO 96/19459), or, pharmaceutically acceptable saltsthereof.

The term “renin inhibitor” includes ditekiren (chemical name:[1S-[1R*,2R*,4R*(1R*,2R*)]]-1-[(1,1-dimethylethoxy)carbonyl]-L-prolyl-L-phenylalanyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2-methyl-1-[[(2-pyridinylmrthyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-N-alfa-methyl-L-histidinamide);terlakiren (chemical name:[R—(R*,S*)]-N-(4-morpholinylcarbonyl)-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2-hydroxy-3-(1-methylethoxy)-3-oxopropyl]-S-methyl-L-cysteineamide);Aliskiren (chemical name:(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide) and zankiren(chemical name: [1S-[1R*[R*(R*)],2S*,3R*]]-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]-alfa-[[2-[[(4-methyl-1-piperazinyl)sulfonyl]methyl]-1-oxo-3-phenylpropyl]-amino]-4-thiazolepropanamide),or, hydrochloride salts thereof, or, SPP630, SPP635 and SPP800 asdeveloped by Speedel, or RO 66-1132 and RO 66-1168 of Formula (A) and(B):

or, pharmaceutically acceptable salts thereof.

The term “aliskiren”, if not defined specifically, is to be understoodboth as the free base and as a salt thereof, especially apharmaceutically acceptable salt thereof, most preferably ahemi-fumarate salt thereof.

The term “calcium channel blocker (CCB)” includes dihydropyridines(DHPs) and non-DHPs (e.g., diltiazem-type and verapamil-type CCBs).Examples include amlodipine, Bepridil, Diltiazem, felodipine, ryosidine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, nitrendipine, Verapamil andnivaldipine, and is preferably a non-DHP representative selected fromthe group consisting of flunarizine, prenylamine, diltiazem, fendiline,gallopamil, mibefradil, anipamil, tiapamil and verapamil, or,pharmaceutically acceptable salts thereof. CCBs may be used asanti-hypertensive, anti-angina pectoris, or anti-arrhythmic drugs.

The term “diuretic” includes thiazide derivatives (e.g., chlorothiazide,hydrochlorothiazide, methylclothiazide, and chlorothalidon).

The term “ApoA-I mimic” includes D4F peptides (e.g., formulaD-W—F—K-A-F—Y-D-K—V-A-E-K—F—K-E-A-F (SEQ ID NO: 21))

An angiotensin II receptor antagonist or a pharmaceutically acceptablesalt thereof is understood to be an active ingredient which bind to theAT₁-receptor subtype of angiotensin II receptor but do not result inactivation of the receptor. As a consequence of the inhibition of theAT₁ receptor, these antagonists can, for example, be employed asantihypertensives or for treating congestive heart failure.

The class of AT₁ receptor antagonists comprises compounds havingdiffering structural features, essentially preferred are thenon-peptidic ones. For example, mention may be made of the compoundswhich are selected from the group consisting of valsartan, losartan,candesartan, eprosartan, irbesartan, saprisartan, tasosartan,telmisartan, the compound with the designation E-1477 of the followingformula

the compound with the designation SC-52458 of the following formula

and the compound with the designation ZD-8731 of the following formula

or, in each case, a pharmaceutically acceptable salt thereof.

Preferred AT₁-receptor antagonist are candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan. Also preferred are thoseagents which have been marketed, most preferred is valsartan or apharmaceutically acceptable salt thereof.

The term “anti-diabetic agent” includes insulin secretion enhancers thatpromote the secretion of insulin from pancreatic-cells. Examples includebiguanide derivatives (e.g., metformin), sulfonylureas (SU) (e.g.,tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrrolidinylamino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-metanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide, and tolylcyclamide), orpharmaceutically acceptable salts thereof. Further examples includephenylalanine derivatives (e.g., nateglinide[N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine] (cf. EP 196222and EP 526171) of the formula

repaglinide[(S)-2-ethoxy-4-{2-[[3-methyl-1-[2-(1-piperidinyl)phenyl]butyl]amino]-2-oxoethyl}benzoicacid] (cf. EP 589874, EP 147850 A2, in particular Example 11 on page 61,and EP 207331 A1); calcium(2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinlycarbonyl)-propionatedihydrate (e.g., mitiglinide (cf. EP 507534)); and glimepiride (cf. EP31058).

Further examples of second agents with which the peptide and polypeptideof the invention can be used in combination include DPP-IV inhibitors,GLP-1 and GLP-1 agonists.

DPP-IV is responsible for inactivating GLP-1. More particularly, DPP-IVgenerates a GLP-1 receptor antagonist and thereby shortens thephysiological response to GLP-1. GLP-1 is a major stimulator ofpancreatic insulin secretion and has direct beneficial effects onglucose disposal.

The DPP-IV (dipeptidyl peptidase IV) inhibitor can be peptidic or,preferably, non-peptidic. DPP-IV inhibitors are in each case genericallyand specifically disclosed e.g. in WO 98/19998, DE 196 16 486 A1, WO00/34241 and WO 95/15309, in each case in particular in the compoundclaims and the final products of the working examples, thesubject-matter of the final products, the pharmaceutical preparationsand the claims are hereby incorporated into the present application byreference to these publications. Preferred are those compounds that arespecifically disclosed in Example 3 of WO 98/19998 and Example 1 of WO00/34241, respectively.

GLP-1 (glucagon like peptide-1) is an insulinotropic protein which isdescribed, e.g., by W. E. Schmidt et al. in Diabetologia, 28, 1985,704-707 and in U.S. Pat. No. 5,705,483.

The term “GLP-1 agonists” includes variants and analogs ofGLP-1(7-36)NH₂ which are disclosed in particular in U.S. Pat. No.5,120,712, U.S. Pat. No. 5,118,666, U.S. Pat. No. 5,512,549, WO 91/11457and by C. Orskov et al in J. Biol. Chem. 264 (1989) 12826. Furtherexamples include GLP-1(7-37), in which compound the carboxy-terminalamide functionality of Arg³⁶ is displaced with Gly at the 37^(th)position of the GLP-1(7-36)NH₂ molecule and variants and analogs thereofincluding GLN⁹-GLP-1(7-37), D-GLN⁹-GLP1-(7-37), acetyl LYS⁹-GLP-1(7-37),LYS¹⁸-GLP-1(7-37) and, in particular, GLP-1(7-37)OH, VAL⁸-GLP-1(7-37),GLY^(8x-GLP-)1(7-37), THR⁸-GLP-1(7-37), MET⁸-GLP-1(7-37) and4-imidazopropionyl-GLP-1. Special preference is also given to the GLPagonist analog exendin-4, described by Greig et al. in Diabetologia1999, 42, 45-50.

Also included in the definition “anti-diabetic agent” are insulinsensitivity enhancers which restore impaired insulin receptor functionto reduce insulin resistance and consequently enhance the insulinsensitivity. Examples include hypoglycemic thiazolidinedione derivatives(e.g., glitazone,(S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione(englitazone),5-{[4-(3-(5-methyl-2-phenyl-4-oxazolyl)-1-oxopropyl)-phenyl]methyl}-thiazolidine-2,4-dione(darglitazone),5-{[4-(1-methyl-cyclohexyl)methoxy)-phenyl]nethyl}-thiazolidine-2,4-dione(ciglitazone),5-{[4-(2-(1-indolyl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(DRF2189),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy)]benzyl}-thiazolidine-2,4-dione(BM-13.1246), 5-(2-naphthylsulfonyl)-thiazolidine-2,4-dione (AY-31637),bis{4-[(2,4-dioxo-5-thiazolidinyl)methyl]phenyl}methane (YM268),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl}-thiazolidine-2,4-dione(AD-5075),5-[4-(1-phenyl-1-cyclopropanecarbonylamino)-benzyl]-thiazolidine-2,4-dione(DN-108)5-{[4-(2-(2,3-dihydroindol-1-yl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione,5-[3-(4-chloro-phenyl])-2-propynyl]-5-phenylsulfonyl)thiazolidine-2,4-dione,5-[3-(4-chlorophenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl)thiazolidine-2,4-dione,5-{[4-(2-(methyl-2-pyridinyl-amino)-ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(rosiglitazone),5-{[4-(2-(5-ethyl-2-pyridyl)ethoxy)phenyl]-methyl}thiazolidine-2,4-dione(pioglitazone),5-{[4-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy)-phenyl]-methyl}-thiazolidine-2,4-dione(troglitazone),5-[6-(2-fluoro-benzyloxy)naphthalen-2-ylmethyl]-thiazolidine-2,4-dione(MCC555),5-{[2-(2-naphthyl)-benzoxazol-5-yl]-methyl}thiazolidine-2,4-dione(T-174) and5-(2,4-dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl-benzyl)benzamide(KRP297)).

Further anti-diabetic agents include, insulin signalling pathwaymodulators, like inhibitors of protein tyrosine phosphatases (PTPases),antidiabetic non-small molecule mimetic compounds and inhibitors ofglutamine-fructose-6-phosphate amidotransferase (GFAT); compoundsinfluencing a dysregulated hepatic glucose production, like inhibitorsof glucose-6-phosphatase (G6Pase), inhibitors offructose-1,6-bisphosphatase (F-1,6-Bpase), inhibitors of glycogenphosphorylase (GP), glucagon receptor antagonists and inhibitors ofphosphoenolpyruvate carboxykinase (PEPCK); pyruvate dehydrogenase kinase(PDHK) inhibitors; inhibitors of gastric emptying; insulin; inhibitorsof GSK-3; retinoid X receptor (RXR) agonists; agonists of Beta-3 AR;agonists of uncoupling proteins (UCPs); non-glitazone type PPARγagonists; dual PPARα/PPARγ agonists; antidiabetic vanadium containingcompounds; incretin hormones, like glucagon-like peptide-1 (GLP-1) andGLP-1 agonists; beta-cell imidazoline receptor antagonists; miglitol;a₂-adrenergic antagonists; and pharmaceutically acceptable saltsthereof.

In one embodiment, the invention provides a combination, in particular apharmaceutical combination, comprising a therapeutically effectiveamount of the polypeptide according to the definition of anyone offormulae I to IV, or an amide, an ester or a salt thereof, and one ormore therapeutically active agents selected from β-adrenergic receptorblockers such as acebutolol, atenolol, betaxolol, bisoprolol,metoprolol, nadolol, propranolol, sotalol and timolol; angiotensin IIreceptor antagonists such as AT1 blockers; antidiabetic agents such asDPPIV inhibitors (e.g. vildagliptin) and GLP1 peptide agonist.

The term “obesity-reducing agent” includes lipase inhibitors (e.g.,orlistat) and appetite suppressants (e.g., sibutramine and phentermine).

An aldosterone synthase inhibitor or a pharmaceutically acceptable saltthereof is understood to be an active ingredient that has the propertyto inhibit the production of aldosterone. Aldosterone synthase (CYP11B2)is a mitochondrial cytochrome P450 enzyme catalyzing the last step ofaldosterone production in the adrenal cortex, i.e., the conversion of11-deoxycorticosterone to aldosterone. The inhibition of the aldosteroneproduction with so-called aldosterone synthase inhibitors is known to bea successful variant to treatment of hypokalemia, hypertension,congestive heart failure, atrial fibrillation or renal failure. Suchaldosterone synthase inhibition activity is readily determined by thoseskilled in the art according to standard assays (e.g., US 2007/0049616).

The class of aldosterone synthase inhibitors comprises both steroidaland non-steroidal aldosterone synthase inhibitors, the later being mostpreferred.

Preference is given to commercially available aldosterone synthaseinhibitors or those aldosterone synthase inhibitors that have beenapproved by the health authorities.

The class of aldosterone synthase inhibitors comprises compounds havingdiffering structural features. An example of non-steroidal aldosteronesynthase inhibitor is the (+)-enantiomer of the hydrochloride offadrozole (U.S. Pat. Nos. 4,617,307 and 4,889,861) of formula

or, if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. inUS2007/0049616, in particular in the compound claims and the finalproducts of the working examples, the subject-matter of the finalproducts, the pharmaceutical preparations and the claims are herebyincorporated into the present application by reference to thispublication. Preferred aldosterone synthase inhibitors suitable for usein the present invention include, without limitation4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-methylbenzonitrile;5-(2-chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid (4-methoxybenzyl)methylamide;4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid butyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;5-(2-Chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 4-fluorobenzyl ester;5-(4-Cyano-2-trifluoromethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid methyl ester;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 2-isopropoxyethyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methylbenzonitrile;4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;3-Fluoro-4-(7-methylene-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)benzonitrile;cis-3-Fluoro-4-[7-(4-fluoro-benzyl)-5,6,7,8-tetrahydro-imidazo[1,5-a]pyridin-5-yl]benzonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrileor in each case, the (R) or (S) enantiomer thereof; or if appropriable,a pharmaceutically acceptable salt thereof.

The term aldosterone synthase inhibitors also include compounds andanalogs disclosed in WO2008/076860, WO2008/076336, WO2008/076862,WO2008/027284, WO2004/046145, WO2004/014914, WO2001/076574.

Furthermore Aldosterone synthase inhibitors also include compounds andanalogs disclosed in U.S. patent applications US2007/0225232,US2007/0208035, US2008/0318978, US2008/0076794, US2009/0012068,US20090048241 and in PCT applications WO2006/005726, WO2006/128853,WO2006128851, WO2006/128852, WO2007065942, WO2007/116099, WO2007/116908,WO2008/119744 and in European patent application EP 1886695. Preferredaldosterone synthase inhibitors suitable for use in the presentinvention include, without limitation8-(4-Fluorophenyl)-5,6-dihydro-8H-imidazo[5,1-c1[1,41oxazine;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-fluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2,6-difluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-methoxybenzonitrile;3-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)phthalonitrile;4-(8-(4-Cyanophenyl)-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)naphthalene-1-carbonitrile;8-[4-(1H-Tetrazol-5-yl)phenyl]-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazineas developed by Speedel or in each case, the (R) or (S) enantiomerthereof; or if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. in WO2009/156462 and WO 2010/130796, in particular in the compound claims andthe final products of the working examples, the subject-matter of thefinal products, the pharmaceutical preparations and the claims.Preferred Aldosterone Synthase inhibitors suitable for combination inthe present invention include,3-(6-Fluoro-3-methyl-2-pyridin-3-yl-1H-indol-1-ylmethyl)-benzonitrilehydrochloride,1-(4-Methanesulfonyl-benzyl)-3-methyl-2-pyridin-3-yl-1H-indole,2-(5-Benzyloxy-pyridin-3-yl)-6-chloro-1-methyl-1H-indole,5-(3-Cyano-1-methyl-1H-indol-2-yl)-nicotinic acid ethyl ester,N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,Pyrrolidine-1-sulfonic acid5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl ester,N-Methyl-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,6-Chloro-1-methyl-2-{5-[(2-pyrrolidin-1-yl-ethylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,6-Chloro-2-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-pyridin-3-yl]-1-methyl-1H-indole-3-carbonitrile,6-Chloro-1-methyl-2-{5-[(1-methyl-piperidin-4-ylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,Morpholine-4-carboxylic acid[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-amide,N-[5-(6-Chloro-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,C,C,C-Trifluoro-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-4-trifluoromethyl-benzenesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-1-phenyl-methanesulfonamide,N-(5-(3-chloro-4-cyanophenyl)pyridin-3-yl)butane-1-sulfonamide,N-(1-(5-(4-cyano-3-methoxyphenyl)pyridin-3-yl)ethyl)ethanesulfonamide,N-((5-(3-chloro-4-cyanophenyppyridin-3-yl)(cyclopropyl)methyl)ethanesulfonamide,N-(cyclopropyl(5-(1H-indol-5-yl)pyridin-3-yl)methyl)ethanesulfonamide,N-(cyclopropyl(5-naphtalen-1-yl-pyridin-3-yl)methyl)ethanesulfonamide,Ethanesulfonic acid[5-(6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-pyridin-3-ylmethyl]-amideand Ethanesulfonic acid{[5-(3-chloro-4-cyano-phenyl)-pyridin-3-yl]-cyclopropyl-methyl}-ethyl-amide.

The term “endothelin receptor blocker” includes bosentan andambrisentan.

The term “CETP inhibitor” refers to a compound that inhibits thecholesteryl ester transfer protein (CETP) mediated transport of variouscholesteryl esters and triglycerides from HDL to LDL and VLDL. Such CETPinhibition activity is readily determined by those skilled in the artaccording to standard assays (e.g., U.S. Pat. No. 6,140,343). Examplesinclude compounds disclosed in U.S. Pat. No. 6,140,343 and U.S. Pat. No.6,197,786 (e.g.,[2R,4q4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylicacid ethyl ester (torcetrapib); compounds disclosed in U.S. Pat. No.6,723,752 (e.g.,(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol);compounds disclosed in U.S. patent application Ser. No. 10/807,838;polypeptide derivatives disclosed in U.S. Pat. No. 5,512,548;rosenonolactone derivatives and phosphate-containing analogs ofcholesteryl ester disclosed in J. Antibiot, 49(8): 815-816 (1996), andBioorg. Med. Chem. Lett; 6:1951-1954 (1996), respectively. Furthermore,the CETP inhibitors also include those disclosed in WO2000/017165,WO2005/095409, WO2005/097806, WO 2007/128568, WO2008/009435, WO2009/059943 and WO2009/071509.

The term “NEP inhibitor” refers to a compound that inhibits neutralendopeptidase (NEP) EC 3.4.24.11. Examples include Candoxatril,Candoxatrilat, Dexecadotril, Ecadotril, Racecadotril, Sampatrilat,Fasidotril, Omapatrilat, Gemopatrilat, Daglutril, SCH-42495, SCH-32615,UK-447841, AVE-0848, PL-37 and(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicacid ethyl ester or a pharmaceutically acceptable salt thereof. NEPinhibitors also include Phosphono/biaryl substituted dipeptidederivatives, as disclosed in U.S. Pat. No. 5,155,100. NEP inhibitorsalso include N-mercaptoacyl phenylalanine derivative as disclosed in PCTapplication Number WO 2003/104200. NEP inhibitors also includedual-acting antihypertensive agents as disclosed in PCT applicationNumbers WO 2008/133896, WO 2009/035543 or WO 2009/134741. Other examplesinclude compounds disclosed in U.S. application Ser. No. 12/788,794;12/788,766 and 12/947,029. NEP inhibitors also include compoundsdisclosed in WO 2010/136474, WO 2010/136493, WO 2011/061271 and U.S.provisional applications No. 61/414,171 and 61/414,163.

In one embodiment, the invention provides a method of activating the APJreceptor in a subject, wherein the method comprises administering to thesubject a therapeutically effective amount of the polypeptide accordingto the definition of anyone of formulae I to IV, or an amide, an esteror a salt thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation of the APJ receptor,in a subject, wherein the method comprises administering to the subjecta therapeutically effective amount of the polypeptide according to thedefinition of anyone of formulae I to IV, or an amide, an ester or asalt thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation (agonism) of the APJreceptor, in a subject, wherein the disorder or the disease is selectedfrom acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

In one embodiment, the invention provides a polypeptide according to thedefinition of anyone of formulae I to IV, for use as a medicament.

In one embodiment, the invention provides the use of a polypeptideaccording to the definition of anyone of formulae I to IV, or an amide,an ester or a salt thereof, in the manufacture of a medicament, for thetreatment of a disorder or disease responsive to the activation of theAPJ receptor. In another embodiment, the invention provides the use of apolypeptide according to the definition of anyone of formulae I to IV,or an amide, an ester or a salt thereof, in the manufacture of amedicament, for the treatment of a disorder or disease responsive to theactivation of the APJ receptor, wherein said disorder or disease is inparticular selected from acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

EXEMPLIFICATION OF THE INVENTION Peptide and Polypeptide Synthesis

Abbreviation Definition AA Amino acid Ac Acetyl Acm Acetamidomethyl ACNAcetonitrile AcOH Acetic acid Ac₂O Acetic anhydride ε-Ahx ε-Aminohexanoic acid AM Aminomethyl BAL Backbone amide linker BSA Bovine SerumAlbumin Boc tert-Butyloxycarbonyl Bzl Benzyl DCM Dichlormethane DICN,N′-Diisopropylcarbodiimide DIPEA N,N′-Diisopropylethylamine DMAN,N′-Dimethylacetamide DMF N,N′-Dimethylformamide DMSO DimethylsulfoxideDTT Dithiothreitol DVB Divinylbenzene EDT Ethanedithiol FA Formic acidFmoc 9-Fluorenylmethyloxycarbonyl HATU2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate HBSS Hank's buffered salt solution HCTU2-(6-Chloro-1H-Benzotriazole-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid HFIP Hexafluoroisopropanol HOAt 1-Hydroxy-7-azabenzotriazole HPLCHigh performance liquid chromatography ivDde(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LN Logarithmusnaturali (natural logarithm) MeOH Methanol MS Mass spectrometry Nal2-Naphthylalanine Nle Norleucine NMP N-Methylpyrrolidine Oxyma PureEthyl 2-cyano-2-(hydroxyimino)acetate Pbf2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl pE Pyroglutamate PhPPhenylproline Pip Pipecolic acid PG Protecting group Ph Phenyl PolPolymer support PS Polystyrene rt Room temperature SPPS Solid phasepeptide synthesis tBuOH tert-Butanol TFA Trifluoroacetic acid THFTetrahydrofuran TIS Triisopropylsilane t_(R) Retention time Trt TritylUPLC Ultra performance liquid chromatography UV Ultraviolet

The peptides were synthesized by standard solid phase Fmoc chemistry.The peptides were assembled on the Prelude™ peptide synthesizer (ProteinTechnologies, Inc., Tucson, USA) and Liberty microwave peptidesynthesizer (CEM Corporation, North Carolina, USA). Peptides with a freecarboxylic acid on the C-terminus were synthesized from 2-chlorotritylchloride-PS-resin (ABCR, Karlsruhe, Germany or AnaSpec, Inc.,California, USA). Peptides with an unsubstituted carboxamide on theC-terminus were synthesized from Fmoc protected Rink-Amide-AM-PS-resin(Merck, Darmstadt, Germany). Peptides with an N-monosubstitutedcarboxamide on the C-terminus were synthesized from BAL-AM-PS-resinloaded with amines (EMC Microcollections, Tubingen, Germany).

The peptides were purified by preparative reversed-phase HPLC. Thefollowing columns were used:

-   -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×100 mm, Part No.        186002572 (one column or two columns in series)    -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×150 mm, Part No.        186002797    -   Waters Atlantis Prep OBD T3 Column, 5 μm, 30×150 mm, Part No.        186003703    -   Waters XBridge Prep C8 OBD Column, 5 μm, 30×150 mm, Part No.        186003083    -   Machery-Nagel Nucleosil® 100-5 C18, 5 μm, 250×40 mm, Part No.        715340.400

Mobile phases consisted of eluent A (0.1% TFA in H₂O) and eluent B(ACN). Gradients were designed based on the specific requirements of theseparation problem. Pure products were lyophilized from ACN/H₂O.

The products were analyzed by analytical HPLC using UV detection atλ=214 nm (Column: Bischoff UHC-640, 53×4.0 mm, ProntoSil 120-3-C18-H, 3μm, Part No. 0604F185PS030 or XBridge BEH300, 100×4.6 mm, C18, 3 μm,Part No. 186003612). Mobile phases consisted of eluent A (0.07% TFA inH₂O) and eluent B (0.1% TFA in ACN) or eluent A (0.1% TFA in H₂O),eluent B (0.4% TFA in ACN), and eluent C (MeOH) or eluent A (0.1% TFA inH₂O), eluent B (0.1% TFA in ACN). Additional characterization of theproducts was done by UPLC-MS or UPLC-HRMS (Column: Waters Acquity UPLC®BEH C18, 1.7 μm, 2.1×50 mm, Part No. 186002350) using electrosprayionization. Mobile phases consisted of eluent A (0.1% FA in H₂O) andeluent B (0.1% FA in ACN) or eluent A (0.05% FA+3.75 mM ammonium acetatein H₂O) and eluent B (0.04% FA in ACN).

The peptides that are exemplified in Table 4 were synthesized using thegeneral procedures described below. Unsubstituted N- or C-termini areindicated by small italic or —OH, respectively.

TABLE 4 SEQ Example Sequence ID NO: Example 1pE-R-P-R-L-a-H-K-G-Pip-nle-a-f-OH 22 Example 2pE-R-P-R-(D-Leu)-S-a-K-G-P-nle-a-f-OH 23 Example 3pE-r-P-R-L-a-H-K-G-P-nle-a-f-OH 24 Example 4pE-R-P-R-(D-Leu)-S-Aib-K-G-P-nle-a-f-OH 25 Example 5pE-R-P-R-L-S-Aib-k-G-P-nle-a-f-OH 26 Example 6pE-R-P-R-L-a-H-K-G-P-nle-f-OH 27 Example 7pE-R-P-R-L-c(tBu)-H-K-G-P-nle-a-f-OH 28 Example 8pE-R-P-R-L-d-H-K-G-P-nle-a-f-OH 29 Example 9pE-R-(trans-4-PhP)-R-L-a-H-K-G-P-nle-a-f-OH 30 Example 10pE-R-P-R-L-(D-Leu)-H-K-G-P-nle-a-f-OH 31 Example 11pE-R-P-R-L-k-H-K-G-P-nle-a-f-OH 32 Example 12pE-R-P-R-L-s-H-K-G-P-nle-a-f-OH 33 Example 13pE-R-P-R-L-a-H-K-G-P-nle-p-f-OH 34 Example 14pE-R-P-R-L-a-H-K-G-P-nle-(D-Leu)-f-OH 35 Example 15pE-R-P-R-L-a-Aib-K-G-P-nle-a-f-OH 36 Example 16pE-R-P-R-L-a-h-K-G-P-nle-a-f-OH 37 Example 17pE-R-P-R-L-a-F-K-G-P-nle-a-f-OH 38 Example 18pE-R-P-R-L-a-H-K-G-P-nle-F-OH 39 Example 19pE-R-P-R-L-a-H-K-G-P-nle-e-f-OH 40 Example 20pE-R-P-R-L-a-H-K-G-P-nle-r-f-OH 41 Example 21pE-R-P-R-L-a-H-K-G-P-nle-a-y-OH 42 Example 22pE-R-P-R-L-a-H-K-G-P-nle-a-d-OH 43 Example 23pE-R-P-R-Cha-a-H-K-G-P-nle-a-f-OH 44 Example 24pE-R-P-R-(3,4-Cl2-F)-a-H-K-G-P-nle-a-f-OH 45 Example 25pE-R-P-R-(2-Nal)-a-H-K-G-P-nle-a-f-OH 46 Example 26pE-R-P-R-L-a-H-K-G-P-nle-a-tic-OH 47 Example 27pE-R-P-R-Y(Bzl)-a-H-K-G-P-nle-a-f-OH 48 Example 28pE-R-P-R-Y-a-H-K-G-P-nle-a-f-OH 49 Example 29pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH 50 Example 30pE-R-P-R-L-a-H-K-G-P-Nle-a-f-OH 51 Example 31pE-R-P-R-L-a-H-k-G-P-Nle-a-f-OH 52 Example 32pE-R-P-R-L-a-H-K-G-P-nle-NH(Phenethyl) 53 Example 33pE-R-P-R-L-a-H-K-G-P-nle-a-f-NH2 54 Example 34pE-R-P-R-L-a-H-K-G-P-Nle-P-f-OH 55 Example 35pE-R-P-R-L-a-H-K-G-P-Nle-f-OH 56 Example 36pE-R-P-R-L-a-H-K-G-P-nle-a-NH(Phenethyl) 57 Example 37pE-R-P-R-L-S-Aib-K-G-P-nle-a-F-OH 58 Example 38pE-R-P-R-L-S-Aib-K-G-P-Nle-a-f-OH 59 Example 39pE-R-P-R-L-a-H-K-G-P-Nle-a-F-OH 60 Example 40pE-R-P-R-L-S-Aib-K-G-P-nle-a-f-OH 61 Example 41pE-R-P-R-L-S-Aib-K-G-P-Nle-P-f-OH 62 Example 42pE-R-P-R-L-S-Aib-K-G-P-Nle-a-F-OH 63 Example 43pE-R-P-R-L-a-H-K-G-P-nle-(4-Phenoxypiperidin- 64 1-yl) Example 44pE-R-P-R-L-abu-H-K-G-P-nle-a-f-OH 65 Example 45pE-R-P-R-L-a-H-K-G-P-nle-abu-f-OH 66 Example 46pE-R-P-R-L-a-f-K-G-P-nle-a-f-OH 67 Example 47pE-R-P-R-L-a-L-K-G-P-nle-a-f-OH 68 Example 48pE-R-P-R-L-a-a-K-G-P-nle-a-f-OH 69 Example 49pE-R-a-R-L-a-H-K-G-P-nle-a-f-OH 70 Example 50H-R-P-R-L-a-H-K-G-P-nle-f-OH 71 Example 51pE-R-P-R-L-S-a-K-G-P-nle-a-f-OH 72 Example 52pE-R-P-R-L-nva-H-K-G-P-nle-a-f-OH 73 Example 53pE-R-P-R-L-a-H-K-G-P-nva-a-f-OH 74 Example 54pE-R-P-R-L-a-H-K-G-P-nle-nva-f-OH 75 Example 55pE-R-P-R-L-S-f-K-G-P-nle-a-f-OH 76 Example 56pE-R-P-R-L-S-h-K-G-P-nle-a-f-OH 77 Example 57pE-R-P-R-L-a-H-K-G-P-f-a-f-OH 78 Example 58pE-R-P-R-L-A-h-K-G-P-nle-a-f-OH 79 Example 59pE-R-P-R-L-a-H-Q-G-P-nle-a-f-OH 80 Example 60pE-R-P-R-L-a-H-E-G-P-nle-a-f-OH 81 Example 61pE-R-P-R-L-v-H-K-G-P-nle-a-f-OH 82 Example 62pE-R-P-R-L-a-H-K-D-P-nle-a-f-OH 83 Example 63pE-R-P-R-Cha-nva-H-K-G-P-nle-a-f-OH 84Analytical Methods1a) HPLC—Analytical Method A

-   -   Column: Bischoff UHC-640 (53×4.0 mm) with ProntoSil 120-3-C18-H,        3 μm; Part n°: 0604F185PS030    -   Eluent A: 0.07% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.5 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 90 10 9.5 0 100 12.0 0 100 12.2 90 101b) HPLC—Analytical Method B

-   -   Column: Bischoff UHC-640 (53×4.0 mm) with ProntoSil 120-3-C18-H,        3 μm; Part n°: 0604F185PS030    -   Eluent A: 0.1% TFA in water/Eluent B: 0.4% TFA in ACN/Eluent C:        MeOH    -   Flow: 1.5 ml/min    -   Temperature: 25° C.    -   Gradient:

Time [min] A [%] B [%] C [%] 0.0 90 2.5 7.5 9.5 0 25 75 12.0 0 25 7512.2 90 2.5 7.51c) HPLC—Analytical Method C

-   -   Column: XBridge BEH300 C18 (100×4.6 mm), 3 μm; Part n°:        186003612    -   Eluent A: 0.1% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 98 2 18 2 98 20 2 98 22 98 22) UPLC-MS—Analytic Method D

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.1% FA in water; Eluent B: 0.1% FA in ACN    -   Flow: 0.7 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 99 1 1.0 97 3 3.5 50 50 4.0 10 90 4.3 0 1004.6 80 203) UPLC-HRMS—Analytic Method E

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n°:        186002350    -   Eluent A: 0.05% FA+3.75 mM ammonium acetate in water; Eluent B:        0.04% FA in ACN    -   Flow: 1.0 ml/min    -   Temperature: 50° C.    -   Gradient: 2 to 98% in 4.4 min        4) HPLC—Analytical Method F    -   Column: YMC-Gel ODS-A-C18    -   Eluent A: 0.1% v/v TFA in Water/Eluent B: ACN/Eluent A, 9/1 v/v    -   Elution with gradient:

Time Flux A B [min] [ml/min] [% v/v] [% v/v] 0 50 95 5 10 50 95 5 40 5080 20 45 50 80 20 55 50 75 25 65 50 75 25 75 50 70 30 105 50 70 30The analytical data for peptides of Examples 1 to 63 are summarized inTable 5 and was generated using the analytical methods described supra.

TABLE 5 HPLC Mass spectrometry t_(R) [M + 2H]²⁺ [M + 3H]³⁺ [M + 2H]²⁺[M + 3H]³⁺ Peptide [min] Meth. (measured) (measured) Meth. (calc.)(calc.) Example 1 3.43 A 496.7 D 744.4 496.6 Example 2 3.40 A 712.3475.1 D 712.4 475.3 Example 3 3.28 A 491.9 D 737.4 491.9 Example 4 3.40A 719.2 479.9 D 719.4 479.9 Example 5 3.44 A 719.2 479.7 D 719.4 479.9Example 6 3.40 A 468.2 D 701.9 468.3 Example 7 3.69 A 521.2 D 781.4521.3 Example 8 3.24 A 506.5 D 759.4 506.6 Example 9 3.65 A 517.2 D775.4 517.3 Example 10 3.58 A 505.7 D 758.4 506.0 Example 11 3.13 A510.8 D 765.9 511.0 Example 12 3.26 A 497.1 D 745.4 497.3 Example 133.43 A 750.2 500.3 D 750.4 500.6 Example 14 3.59 A 505.7 D 758.4 506.0Example 15 3.57 A 711.1 474.5 D 711.4 474.6 Example 16 3.40 A 491.7 D737.4 491.9 Example 17 3.88 A 742.2 495.1 D 742.4 495.3 Example 18 3.43A 468.1 D 701.9 468.3 Example 19 3.27 A 766.4 D 766.4 511.3 Example 203.02 A 520.2 D 779.9 520.3 Example 21 2.82 A 745.3 D 745.4 497.3 Example22 2.49 A 481.2 D 721.4 481.3 Example 23 3.52 A 505.2 D 757.4 505.3Example 24 3.65 A 526.0 D 788.4 525.9 Example 25 3.57 A 779.4 519.8 D779.4 519.9 Example 26 3.24 A 495.9 D 743.4 496.0 Example 27 3.89 A807.3 D 807.4 538.6 Example 28 3.12 A 508.5 D 762.4 508.6 Example 293.35 A 491.8 D 737.4 491.9 Example 30 5.03 B 491.7 D 737.4 491.9 Example31 5.22 B 737.4 491.9 D 737.4 491.9 Example 32 5.47 B 679.9 453.6 D679.8 453.6 Example 33 3.17 A 736.7 491.6 D 736.9 491.6 Example 34 3.37A 500.6 D 750.4 500.6 Example 35 3.28 A 468.2 D 701.9 468.3 Example 363.41 A 477.2 D 715.4 477.3 Example 37 3.59 A 719.3 479.9 D 719.4 479.9Example 38 3.41 A 719.4 479.9 D 719.4 479.9 Example 39 3.27 A 491.9 D737.4 491.9 Example 40 3.48 A 719.2 479.9 D 719.4 479.9 Example 41 3.53A 732.4 488.6 D 732.4 488.6 Example 42 3.49 A 719.3 479.9 D 719.4 479.9Example 43 3.93 A 708.6 472.6 D 707.9 472.3 Example 44 7.04 C 744.4496.6 E 744.9 496.9 Example 45 7.17 C 744.4 496.6 E 744.9 496.9 Example46 7.95 C 742.4 495.3 E 742.9 495.6 Example 47 7.98 C 725.4 484.0 E725.9 484.2 Example 48 7.52 C 704.4 470.0 E 704.8 470.2 Example 49 7.04C 724.4 483.3 E 724.8 483.6 Example 50 6.77 C 646.4 431.3 E 646.8 431.5Example 51 6.79 C 712.4 475.3 E 712.8 475.6 Example 52 7.49 C 751.4501.3 E 751.9 501.6 Example 53 6.46 C 730.4 487.3 E 730.8 487.6 Example54 7.50 C 751.4 501.3 E 751.9 501.6 Example 55 7.67 C 750.4 500.6 E750.9 500.9 Example 56 6.93 C 745.4 497.3 E 745.9 497.6 Example 57 7.16C 754.4 503.3 E 754.9 503.6 Example 58 7.01 C 737.4 492.0 E 737.9 492.2Example 59 7.37 C 737.4 492.0 E 737.9 492.2 Example 60 7.42 C 737.9492.3 E 738.3 492.6 Example 61 7.33 C 751.4 501.3 E 751.9 501.6 Example62 7.33 C 766.4 511.3 E 766.9 511.6 Example 63 8.16 C 771.5 514.6 E771.9 514.9General Synthesis Procedures1) Loading of First Amino Acid onto 2-Chlorotrityl Chloride Resin andFmoc-Removal

2-Chlorotrityl chloride resin (1 eq., 1.0-1.6 mmol/g) was washedthoroughly with DCM. The desired amino acid (typically 0.5-2 eq.relative to the resin, considering 1.6 mmol/g loading) was dissolved inDCM (approx. 10 mL per gram of resin) and DIPEA (4 eq. relative to theresin, considering 1.6 mmol/g loading). The solution was added to theresin and the suspension was shaken at it for 19 h. The resin wasdrained and then thoroughly washed sequentially with DCM/MeOH/DIPEA(17:2:1), DCM, DMA, DCM.

For Fmoc removal and determination of the loading the resin was shakenrepeatedly with piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4)(12×10 mL per gram of initial resin) and washed with DMA (2×10 mL pergram of initial resin). The combined solutions were diluted with MeOH toa volume V of 250 mL per gram of initial resin. A 2 mL aliquot (V_(a))of this solution was diluted further to 250 mL (V_(t)) with MeOH. The UVabsorption was measured at 299.8 nm against a reference of MeOH, givingabsorption A. The resin was thoroughly washed sequentially with DMA,DCM, DMA, DCM and dried in high vacuum at 40° C., affording m g ofresin.

The loading of the resin is calculated according to the formula:Loading [mol/g]=(A×V _(t) ×V)/d×ε×V _(a) ×m)(with d: width of cuvette; ε=7800 L mol⁻¹ cm⁻¹)2) Solid Phase Peptide Synthesis2a) Synthesis Cycle a on Prelude™ Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith 4-methylpiperidine/DMA (1:4). The resin was washed with DMA.Coupling was done by addition of the Fmoc-amino acid (3 eq.; 0.2 Msolution in NMP), HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (3.3eq.; 0.66 M solution in NMP) followed by mixing of the suspension withnitrogen at it for typically 15 min to 4 h depending on the specificrequirements. After washing with DMA the coupling step was typicallyrepeated 1 to 3 times depending on the specific requirements. Afterwashing with DMA capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2b) Synthesis Cycle B on Prelude™ Synthesizer

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4). The resin was washed with DMA. Coupling wasdone by addition of the Fmoc-amino acid (3 eq.; 0.3 M solution in NMP),HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (4.5 eq.; 0.9 M solutionin NMP) followed by mixing of the suspension with nitrogen at rt fortypically 15 min to 4 h depending on the specific requirements. Afterwashing with DMA the coupling step was typically repeated 1 to 3 timesdepending on the specific requirements. After washing with DMA cappingwas performed by addition of a mixture of Ac₂O/pyridine/DMA (1:1:8) andsubsequent mixing of the suspension at rt. The resin was washed withDMA.

2c) Synthesis Cycle C on Liberty™ Synthesizer

The resin was washed with DMF and DCM. Fmoc was removed by treatmentwith 20% piperidine/DMF (typically 7 ml per 0.1 mmol twice). The resinwas washed with DMF and Coupling was done by addition of the Fmoc-aminoacid (5 eq.; 0.2 M solution in DMF), HCTU (5 eq.; 0.5 M solution inDMF), and DIPEA (10 eq.; 2 M solution in NMP) followed by mixing of thesuspension with nitrogen at 75 or 50° C. for typically 5 to 50 min withmicrowave power 0 to 20 watts depending on the specific requirements.After washing with DMF the coupling step might be repeated oncedepending on the specific requirements. The resin was washed with DMF.

3) Cleavage from Resin with or without Concomitant Removal of ProtectingGroups

3a) Cleavage Method A

The resin (0.1 mmol) was shaken at rt for typically 1.5-2 h with 95% aq.TFA/EDT/TIS (95:2.5:2.5) (2 mL). The cleavage solution was filtered off,and fresh solution was added (2 mL). The suspension was shaken at rt fortypically 0.75-1 h then the cleavage solution was filtered off. Freshsolution was added (2 mL) and the suspension was shaken at rt fortypically 0.75-1 h. The cleavage solution was filtered off. The resinwas rinsed once with 95% aq. TFA (1 mL). The combined cleavage andwashing solutions were poured slowly onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum.

3b) Cleavage Method B

The resin (0.1 mmol) was shaken at rt for 1.5 h with 95% aq. TFA/TIS(97.5:2.5) (2 mL). The cleavage solution was filtered off, and freshsolution was added (2 mL). The suspension was shaken at rt for 45 minthen the cleavage solution was filtered off. Fresh solution was added (2mL) and the suspension was shaken at rt for 45 min. The cleavagesolution was filtered off. The resin was rinsed once with 95% aq. TFA (1mL). The combined cleavage and washing solutions were poured slowly ontoa mixture of cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (10mL), the suspension was centrifuged and the supernatant was poured off.The solid was dried in high vacuum.

3c) Cleavage Method C

HFIP/DCM (10:90) (2 mL) was added to the resin (0.1 mmol) and thesuspension was shaken at rt for 10 min. The cleavage solution wasfiltered off into iPrOH (0.8 mL). This step was repeated 3 times,combining the cleavage solutions with the first iPrOH-containingcleavage solution directly. The combined cleavage solutions wereconcentrated to dryness in high vacuum. The residue was lyophilized fromtBuOH/H₂O (4:1).

3d) Cleavage Method D

The resin (0.1 mmol) was shaken at rt for 3 h with 95% aq. TFA/TIS/DTT(95:2.5:2.5) (3 mL). The cleavage solution was filtered off. The resinwas rinsed once with 95% aq. TFA (1 mL). The combined cleavage andwashing solutions were poured slowly onto a mixture of coldheptane/diethyl ether (1:1) (10-15 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. Diethyl ether(10 mL) was added to the residue, the suspension was vortexed for 3 minand centrifuged, and the supernatant was poured off, The wash processwas repeated twice. The solid was dried in high vacuum.

In the following the syntheses of representative examples are described.

Example 1

Synthesis of pE-R—P—R-L-a-H—K-G-Pip-nle-a-f-OH (SEQ ID NO: 22) Example 1below discloses SEQ ID NOS 85 and 22, respectively, in order ofappearance.

Example 1 Preparation of Intermediate 1a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-f-OH, Fmoc Removaland Determination Of the Loading of the Resin)

2-Chlorotrityl chloride resin (3.0 g, 4.80 mmol) was reacted with asolution of Fmoc-f-OH (1.86 g, 4.80 mmol) in DCM (30 mL) and DIPEA (3.35mL, 19.2 mmol) in analogy to the general procedure described above togive Intermediate 1a (3.53 g, loading=0.96 mmol/g).

Preparation of Intermediate 1b

(Assembly of Linear Peptide)

Intermediate 1a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows (SEQ ID NO: 86):

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 a 2 ×15 min A 2 nle 2 × 15 min A 3 Pip 2 × 15 min A 4 G 2 × 30 min A 5 K(Boc)2 × 15 min A 6 H(Trt) 2 × 15 min A 7 a 2 × 15 min A 8 L 2 × 15 min A 9R(Pbf) 4 × 1 h A 10 P 2 × 15 min A 11 R(Pbf) 4 × 1 h A 12 pE 2 × 15 minA

Preparation of Example 1 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added toIntermediate 1b (0.1 mmol) and the suspension was shaken at rt for 1.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at rt for 45 min then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at it for 45 min. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage and washing solutions were poured onto a mixture ofcold heptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 1 (86.5 mg, 0.044 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.43 min) and UPLC-MS (Analytical method C; measured:[M+3H]³⁺=496.7; calculated: [M+3H]³⁺=496.6).

Example 29

Synthesis pE-R—P—R-L-a-H—K-G-P-nle-a-f-OH(SEQ ID NO: 50) Example 29below discloses SEQ ID NOS 87 and 50, respectively, in order ofappearance.

Example 29 Preparation of Intermediate 29a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-f-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (2 g, 3.20 mmol) was reacted with asolution of Fmoc-f-OH (992 mg, 2.56 mmol) in DCM (20 mL) and DIPEA(2.236 mL, 12.80 mmol) in analogy to the general procedure describedabove to give Intermediate 29a (2.36 g, loading=0.82 mmol/g).

Preparation of Intermediate 29b

(Assembly of Linear Peptide)

Intermediate 29a (0.600 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows (SEQ ID NO: 88):

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 a 2 ×15 min B 2 nle 2 × 15 min B 3 P 2 × 15 min B 4 G 2 × 30 min B 5 K(Boc) 2× 15 min B 6 H(Trt) 2 × 15 min B 7 a 2 × 15 min B 8 L 2 × 15 min B 9R(Pbf) 4 × 1 h B 10 P 2 × 15 min B 11 R(Pbf) 4 × 1 h B 12 pE 2 × 15 minB

Preparation of Example 29

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (10 mL) was added toIntermediate 29b (0.6 mmol) and the suspension was shaken at it for 2 h.The cleavage solution was filtered off, and fresh cleavage solution (10mL) was added. The suspension was shaken at it for 1 h then the cleavagesolution was filtered off. Fresh solution (10 mL) was added and thesuspension was shaken at it for 1 h. The cleavage solution was filteredoff. The combined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (200 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (100 mL), thesuspension was centrifuged and the supernatant was poured off. The solidwas dried in high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 29 (538.7 mg, 0.279 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.35 min) and UPLC-MS (Analytical method C; measured:[M+3H]³⁺=491.8; calculated: [M+3H]³⁺=491.9).

Example 32

Synthesis of pE-R—P—R-L-a-H—K-G-P-nle-NH(Phenethyl) (SEQ ID NO: 53)Example 32 below discloses SEQ ID NOS 89 and 53, respectively, in orderof appearance.

Example 32 Preparation of Intermediate 32a

(Assembly of Linear Peptide)

Phenethylamine-BAL-PS resin (167 mg, 0.100 mmol) was subjected to solidphase peptide synthesis on the Prelude™ peptide synthesizer. Couplingwas performed as follows (SEQ ID NO: 90):

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 nle 2× 30 min B 2 P 2 × 30 min B 3 G 3 × 1 h B 4 K(Boc) 2 × 30 min B 5 H(Trt)2 × 30 min B 6 a 2 × 30 min B 7 L 2 × 30 min B 8 R(Pbf) 4 × 1 h B 9 P 2× 90 min B 10 R(Pbf) 4 × 1 h B 11 pE 2 × 90 min B

Preparation of Example 32 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added toIntermediate 32a (0.1 mmol) and the suspension was shaken at rt for 1.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at it for 1.5 h then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at it for 2 h. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (5 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 32 (37.0 mg, 0.020 mmol).

The pure product was analyzed by analytical HPLC (Analytical method B:t_(R)=5.447 min) and UPLC-MS (Analytical method C; measured:[M+3H]³⁺=453.6; calculated: [M+3H]³⁺=453.6).

Example 33

Synthesis of pE-R—P—R-L-a-H—K-G-P-nle-a-f-NH2 (SEQ ID NO: 54) Example 33below discloses SEQ ID NOS 91 and 54, respectively, in order ofappearance.

Example 33 Preparation of Intermediate 33a

(Assembly of Linear Peptide)

Fmoc protected Rink-Amide-AM-PS-resin (217 mg, 0.100 mmol) was subjectedto solid phase peptide synthesis on the Prelude™ peptide synthesizer.Coupling was performed as follows (SEQ ID NO: 92):

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 f 2 ×15 min A 2 a 2 × 1 h A 3 nle 2 × 15 min A 4 P 2 × 15 min A 5 G 2 × 30min A 6 K(Boc) 2 × 15 min A 7 H(Trt) 2 × 15 min A 8 a 2 × 15 min A 9 L 2× 15 min A 10 R(Pbf) 4 × 1 h A 11 P 2 × 30 min A 12 R(Pbf) 4 × 1 h A 13pE 2 × 30 min A

Preparation of Example 33 Cleavage from the Resin with ConcomitantProtecting Group Removal then Purification

A mixture of 95% aq. TFA/TIS (97.5:2.5) (2 mL) was added to Intermediate33a (0.1 mmol) and the suspension was shaken at rt for 1.5 h. Thecleavage solution was filtered off, and fresh cleavage solution (2 mL)was added. The suspension was shaken at rt for 45 min then the cleavagesolution was filtered off. Fresh solution (2 mL) was added and thesuspension was shaken at rt for 45 min. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Example 33 (108.9 mg, 0.055 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.17 min) and UPLC-MS (Analytical method C; measured:[M+3H]³⁺=491.6; calculated: [M+3H]³⁺=491.6).

Example 43

Synthesis pE-R—P—R-L-a-H—K-G-P-nle-(4-Phenoxypiperidinyl) SEQ ID NO: 64)Example 43 below discloses SEQ ID NOS 93-95 and 64, respectively, inorder of appearance.

Example 43 Preparation of Intermediate 43a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-nle-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (300 mg, 0.48 mmol) was reacted with asolution of Fmoc-Nle-OH (136 mg, 0.384 mmol) in DCM (3 mL) and DIPEA(0.335 mL, 1.92 mmol) in analogy to the general procedure describedabove to give Intermediate 43a (329 mg, loading=0.99 mmol/g).

Preparation of Intermediate 43b

(Assembly of Linear Peptide)

Intermediate 43a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows (SEQ ID NO: 96):

Number of couplings × Coupling AA Reaction time Synthesis cycle 1 P 2 ×15 min A 2 G 2 × 30 min A 3 K(Boc) 2 × 15 min A 4 H(Trt) 2 × 15 min A 5a 2 × 15 min A 6 L 2 × 15 min A 7 R(Pbf) 4 × 1 h A 8 P 2 × 30 min A 9R(Pbf) 4 × 1 h A 10 pE 2 × 30 min A

Preparation of Intermediate 43c

(HFIP Cleavage from the Resin)

HFIP/DCM (10:90) (2 mL) was added to Intermediate 43b (0.100 mmol) andthe suspension was stirred at it for 10 min. The cleavage solution wasfiltered off into iPrOH (0.8 mL). This step was repeated 3 times,combining the cleavage solutions with the first iPrOH-containingcleavage solution directly. The combined cleavage solutions wereconcentrated to dryness in high vacuum. The residue was lyophilized fromtBuOH/H₂O (4:1) to give Intermediate 43c.

Preparation of Intermediate 43d

(Coupling of 4-phenoxypiperidine)

A mixture of Intermediate 43c with HATU (49.4 mg, 0.130 mmol), HOAt(17.7 mg, 0.130 mmol) and 2,6-lutidine (0.233 mL, 2.000 mmol) in NMP (5mL) was stirred at rt for 5 min. 4-Phenoxypiperidine (35.0 mg, 0.197mmol) was added and stirring was continued at rt for 45 min. Thereaction mixture was concentrated to dryness in vacuo to giveIntermediate 43d.

Preparation of Example 43

(Removal of Protecting Groups and Purification)

Intermediate 43d was dissolved in 95% aq. TFA/EDT/TIS (95:2.5:2.5) (5mL) and the solution was stirred at rt for 2 h. The cleavage solutionwas poured onto cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (5mL), the suspension was centrifuged and the supernatant poured off. Theresidue was dried in high vacuum. The product was isolated bypreparative HPLC and lyophilized from ACN/H₂O to afford Example 43 (19.3mg, 0.010 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.93 min) and UPLC-MS (Analytical method C; measured:[M+3H]³⁺=472.6; calculated: [M+3H]³⁺=472.3).

Example 57

Synthesis of pE-R—P—R-L-a-H—K-G-P-f-a-f-OH (SEQ ID NO: 78) Example 57below discloses SEQ ID NOS 97 and 78, respectively, in order ofappearance.

Preparation of Intermediate 57a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-f-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (5.0 g, 8.01 mmol) was reacted with asolution of Fmoc-f-OH (3.10 g, 8.01 mmol) in DCM (50 mL) and DIPEA (5.59mL, 32.0 mmol) in analogy to the general procedure described above togive Intermediate 57a (5.87 g, loading=0.897 mmol/g).

Preparation of Intermediate 57b

(Assembly of Linear Peptide)

Intermediate 57a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Liberty™ microwave peptide synthesizer. Coupling wasperformed as follows (SEQ ID NO: 98):

Number of couplings × Tem- Micro- Reaction perature wave SynthesisCoupling AA time ° C. power cycle 1 a 1 × 7.5 min 50 20 C 2 f 1 × 7.5min 50 20 C 3 P 1 × 7.5 min 50 20 C 4 G 1 × 7.5 min 50 20 C 5 K(Boc) 1 ×7.5 min 50 20 C 6 H(Trt) 1 × 2 min 50 0 C 1 × 4 min 50 25 7 a 1 × 7.5min 50 25 C 8 L 1 × 7.5 min 50 25 C 9 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5min 50 25 10 P 1 × 7.5 min 50 25 C 11 R(Pbf) 2 × 42 min 50 0 C 2 × 7.5min 50 25 12 pE 1 × 7.5 min 50 25 C

Preparation of Example 57

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A mixture of 95% aq. TFA/EDT/DTT (95:2.5:2.5) (3 mL) was added toIntermediate 57b (0.1 mmol) and the suspension was shaken at it for 3 h.The cleavage solution was filtered off and the resin was washed with 95%aq. TFA (1 mL). The combined cleavage and washing solutions were pouredonto a mixture of cold heptane/diethyl ether (1:1) (11 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. Diethyl ether (10 mL) was added to the residue, the suspension wasvortexed for 3 min and centrifuged, and the supernatant was poured off,The wash process was repeated twice. The solid was dried in high vacuumThe crude was purified by preparative HPLC and lyophilized from ACN/H₂Oto afford Example 57 (59 mg, 0.030 mmol).

The pure product was analyzed by analytical HPLC (Analytical method C:t_(R)=7.16 min) and UPLC-MS (Analytical method E; measured:[M+3H]³⁺=503.3; calculated: [M+3H]³⁺=503.6).

Alternatively, the crude was purified by preparative HPLC (AnalyticalMethod F) and the desired product was isolated as a TFA salt vialyophilization.

This crude isolated TFA salt was dissolved in water (15mL water/mmol ofpolypeptide) and was converted into the acetate salt using an ionexchanger resin of the type strong base in its hydroxide form [Ionexchanger III, strong base, (OH form. 0.9 mmol/ml), Merck-Darmstadt,Germany, Catalog number; 1.04767.0500, (25 eq)], and the pure acetatesalt product was isolated via lyophilization.

The salt stoichiometry was evaluated based on the analysis of the aceticcontent (ion chromatography) and water content and was determined torange between 1:3 and 1:4 (polypeptide:acetate).

The other examples were synthesized in analogy:

-   -   Examples 2 to 28 were synthesized in analogy to Example 1.    -   Example 30 and 31 were synthesized in analogy to Example 29.    -   Example 34 to 42 were synthesized in analogy to Example 33.    -   Example 44 to 56 and 58 to 63 were synthesized in analogy to        Example 57.

The polypeptide in the examples below have been found to have EC₅₀values in the range of about 0.01 nM to about 1100 nM for APJ receptorpotency. The polypeptides in the examples below have been found to havea plasma stability higher than 2 minutes, higher than 5 minutes, higherthan 10 minutes, higher than 20 minutes, higher than 50 minutes andhigher than 60 minutes.

It can be seen that the polypeptides of the invention are useful asagonist of the APJ receptor and therefore useful in the treatment ofdiseases and conditions responsive to the activation of the APJreceptor, such as the diseases disclosed herein.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

What is claimed is:
 1. A polypeptide having the following formula (SEQID NO: 1):X1-X2-X3-R—X5-X6-X7-X8-X9-X10-X11-X12-X13  I wherein: X1 is theN-terminus of the polypeptide and is either absent or pE; X2 is R or r;X3 is P or 4-PhP; X5 is L, Cha, D-L, F, Y, Y(Bzl), 3,4-Cl2-F or Nal; X6is a D-amino acid, S or A; X7 is a D-amino acid, L, H or Aib; and atleast one of X6 and X7 is D-amino acid or Aib; X8 is K, k, Q or E; X9 isG or D; X10 is P or pipecolic acid; X11 is D-Nle, Nle, f or D-Nva; X12is absent, P or a D-amino acid; X13 is the C-terminus and is absent, For a D-amino acid; and at least one of X11, X12 and X13 is a D-aminoacid; wherein: Nle is L-norleucine; D-Nle is D-norleucine; Nal isL-naphthyl)alanine; D-Nva is D-norvaline; Aib is α-aminoisobutyric acid;Cha is (S)-β-cyclohexylalanine; D-Tic isD-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; pE is L-pyroglutamicacid; 3,4-Cl2-F is (S)-3,4-dichlorophenylalanine; Y is L-tyrosine; andY(Bzl) is L-benzyl-tyrosine; or an amide, an ester or a salt of thepolypeptide.
 2. The polypeptide of claim 1 having sequence of (SEQ IDNO: 3) wherein X6 and X12 are D-amino acids; or a salt thereof.
 3. Thepolypeptide of claim 2 having the sequence of (SEQ ID NO: 4) wherein X13is D-amino acid; or a salt thereof.
 4. The polypeptide of claim 3 havingsequence of (SEQ ID NO: 5) wherein X11 is D-amino acid; of a saltthereof.
 5. The polypeptide of claim 1 having sequence of (SEQ ID NO: 6)wherein X6 and X13 are D-amino acids; or a salt thereof.
 6. Apolypeptide according to claim 1 having the following formula (SEQ IDNO: 9):X1-R—P—R—X5-a-X7-X8-G-P—X11-X12-X13  II or a salt of the polypeptide. 7.The polypeptide according to claim 1 having Formula III (SEQ ID NO: 10):X1-R—P—R—X5-X6-X7-K-G-P—X11-a-f  III; or a salt of the polypeptide. 8.The polypeptide according to claim 1 having Formula IV (SEQ ID NO: 11):X1-R—P—R—X5-S—X7-K-G-P—X11-X12-X13  IV; or a salt of the polypeptide. 9.The polypeptide according to claim 1 having sequence of (SEQ ID NO: 12)wherein X6 is a D-amino acid selected from a, D-Leu, k, s, d, nva, abu,f, h, v and D-Cys(tBu); or a salt thereof.
 10. The polypeptide of claim1 having sequence of (SEQ ID NO: 14) wherein X12 is absent or a D-aminoacid selected from a, f, p, e, r, abu, nva, and D-Leu; or a saltthereof.
 11. The polypeptide of claim 1 having sequence of (SEQ ID NO:15) wherein X13 is absent or is a D-amino acid selected from f, y, d,and D-Tic, or a salt thereof.
 12. The polypeptide according to claim 11having sequence of (SEQ ID NO: 16) wherein X13 is absent or f; or a saltof the polypeptide.
 13. The polypeptide according to claim 1 havingsequence of (SEQ ID NO: 17) wherein X1 is pE; or a salt of thepolypeptide.
 14. The polypeptide according to claim 1 having sequence of(SEQ ID NO: 18) wherein X5 is L; or a salt of the polypeptide.
 15. Thepolypeptide according to claim 1 having sequence of (SEQ ID NO: 19)wherein X8 is K; or a salt of the polypeptide.
 16. The polypeptideaccording to claim 1 selected from: (SEQ ID NO: 22)pE-R-P-R-L-a-H-K-G-Pip-nle-a-f-OH (SEQ ID NO: 23)pE-R-P-R-(D-Leu)-S-a-K-G-P-nle-a-f-OH (SEQ ID NO: 24)pE-r-P-R-L-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 25)pE-R-P-R-(D-Leu)-S-Aib-K-G-P-nle-a-f-OH (SEQ ID NO: 26)pE-R-P-R-L-S-Aib-k-G-P-nle-a-f-OH (SEQ ID NO: 27)pE-R-P-R-L-a-H-K-G-P-nle-f-OH (SEQ ID NO: 28)pE-R-P-R-L-c(tBu)-H-K-G-P-nle-a-f-OH (SEQ ID NO: 29)pE-R-P-R-L-d-H-K-G-P-nle-a-f-OH (SEQ ID NO: 30)pE-R-(trans-4-PhP)-R-L-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 31)pE-R-P-R-L-(D-Leu)-H-K-G-P-nle-a-f-OH (SEQ ID NO: 32)pE-R-P-R-L-k-H-K-G-P-nle-a-f-OH (SEQ ID NO: 33)pE-R-P-R-L-s-H-K-G-P-nle-a-f-OH (SEQ ID NO: 34)pE-R-P-R-L-a-H-K-G-P-nle-p-f-OH (SEQ ID NO: 35)pE-R-P-R-L-a-H-K-G-P-nle-(D-Leu)-f-OH (SEQ ID NO: 36)pE-R-P-R-L-a-Aib-K-G-P-nle-a-f-OH (SEQ ID NO: 37)pE-R-P-R-L-a-h-K-G-P-nle-a-f-OH (SEQ ID NO: 38)pE-R-P-R-L-a-F-K-G-P-nle-a-f-OH (SEQ ID NO: 39)pE-R-P-R-L-a-H-K-G-P-nle-F-OH (SEQ ID NO: 40)pE-R-P-R-L-a-H-K-G-P-nle-e-f-OH (SEQ ID NO: 41)pE-R-P-R-L-a-H-K-G-P-nle-r-f-OH (SEQ ID NO: 42)pE-R-P-R-L-a-H-K-G-P-nle-a-y-OH (SEQ ID NO: 43)pE-R-P-R-L-a-H-K-G-P-nle-a-d-OH (SEQ ID NO: 44)pE-R-P-R-Cha-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 45)pE-R-P-R-(3,4-Cl2-F)-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 46)pE-R-P-R-(2-Nal)-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 47)pE-R-P-R-L-a-H-K-G-P-nle-a-tic-OH (SEQ ID NO: 48)pE-R-P-R-Y(Bzl)-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 49)pE-R-P-R-Y-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 50)pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 51)pE-R-P-R-L-a-H-K-G-P-Nle-a-f-OH (SEQ ID NO: 52)pE-R-P-R-L-a-H-k-G-P-Nle-a-f-OH (SEQ ID NO: 53)pE-R-P-R-L-a-H-K-G-P-nle-NH(Phenethyl) (SEQ ID NO: 54)pE-R-P-R-L-a-H-K-G-P-nle-a-f-NH2 (SEQ ID NO: 55)pE-R-P-R-L-a-H-K-G-P-Nle-P-f-OH (SEQ ID NO: 56)pE-R-P-R-L-a-H-K-G-P-Nle-f-OH (SEQ ID NO: 57)pE-R-P-R-L-a-H-K-G-P-nle-a-NH(Phenethyl) (SEQ ID NO: 58)pE-R-P-R-L-S-Aib-K-G-P-nle-a-F-OH (SEQ ID NO: 59)pE-R-P-R-L-S-Aib-K-G-P-Nle-a-f-OH (SEQ ID NO: 60)pE-R-P-R-L-a-H-K-G-P-Nle-a-F-OH (SEQ ID NO: 61)pE-R-P-R-L-S-Aib-K-G-P-nle-a-f-OH (SEQ ID NO: 62)pE-R-P-R-L-S-Aib-K-G-P-Nle-P-f-OH (SEQ ID NO: 63)pE-R-P-R-L-S-Aib-K-G-P-Nle-a-F-OH (SEQ ID NO: 64)pE-R-P-R-L-a-H-K-G-P-nle-(4-Phenoxypiperidin-1-yl) (SEQ ID NO: 65)pE-R-P-R-L-abu-H-K-G-P-nle-a-f-OH (SEQ ID NO: 66)pE-R-P-R-L-a-H-K-G-P-nle-abu-f-OH (SEQ ID NO: 67)pE-R-P-R-L-a-f-K-G-P-nle-a-f-OH (SEQ ID NO: 68)pE-R-P-R-L-a-L-K-G-P-nle-a-f-OH (SEQ ID NO: 69)pE-R-P-R-L-a-a-K-G-P-nle-a-f-OH (SEQ ID NO: 70)pE-R-a-R-L-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 71)H-R-P-R-L-a-H-K-G-P-nle-f-OH (SEQ ID NO: 72)pE-R-P-R-L-S-a-K-G-P-nle-a-f-OH (SEQ ID NO: 73)pE-R-P-R-L-nva-H-K-G-P-nle-a-f-OH (SEQ ID NO: 74)pE-R-P-R-L-a-H-K-G-P-nya-a-f-OH (SEQ ID NO: 75)pE-R-P-R-L-a-H-K-G-P-nle-nva-f-OH (SEQ ID NO: 76)pE-R-P-R-L-S-f-K-G-P-nle-a-f-OH (SEQ ID NO: 77)pE-R-P-R-L-S-h-K-G-P-nle-a-f-OH (SEQ ID NO: 78)pE-R-P-R-L-a-H-K-G-P-f-a-f-OH (SEQ ID NO: 79)pE-R-P-R-L-A-h-K-G-P-nle-a-f-OH (SEQ ID NO: 80)pE-R-P-R-L-a-H-Q-G-P-nle-a-f-OH (SEQ ID NO: 81)pE-R-P-R-L-a-H-E-G-P-nle-a-f-OH (SEQ ID NO: 82)pE-R-P-R-L-v-H-K-G-P-nle-a-f-OH (SEQ ID NO: 83)pE-R-P-R-L-a-H-K-D-P-nle-a-f-OH; (SEQ ID NO: 84)pE-R-P-R-Cha-nva-H-K-G-P-nle-a-f-OH

or an amide, an ester or a salt of the polypeptide.
 17. The polypeptideof claim 16 selected from: (SEQ ID NO: 50)pE-R-P-R-L-a-H-K-G-P-nle-a-f-OH (SEQ ID NO: 22)pE-R-P-R-L-a-H-K-G-Pip-n le-a-f-OH (SEQ ID NO: 25)pE-R-P-R-(D-Leu)-S-Aib-K-G-P-nle-a-f-OH (SEQ ID NO: 51)pE-R-P-R-L-a-H-K-G-P-Nle-a-f-OH (SEQ ID NO: 27)pE-R-P-R-L-a-H-K-G-P-nle-f-OH (SEQ ID NO: 29)pE-R-P-R-L-d-H-K-G-P-nle-a-f-OH (SEQ ID NO: 35)pE-R-P-R-L-a-H-K-G-P-nle-(D-Leu)-f-OH (SEQ ID NO: 36)pE-R-P-R-L-a-Aib-K-G-P-nle-a-f-OH (SEQ ID NO: 37)pE-R-P-R-L-a-h-K-G-P-nle-a-f-OH (SEQ ID NO: 38)pE-R-P-R-L-a-F-K-G-P-nle-a-f-OH; (SEQ ID NO: 78)pE-R-P-R-L-a-H-K-G-P-f-a-f-OH;

or an amide, an ester or a salt of the polypeptide.
 18. The polypeptideaccording to claim 1 wherein the polypeptide has a plasma stability ofat least 100 minutes and an IC50 of less than 10 nM.
 19. A method oftreating or preventing a disease or disorder responsive to the agonismof the APJ receptor, in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of thepolypeptide according to claim 1 or an amide, an ester or a saltthereof.
 20. The method of claim 19 wherein the disease or disorder isselected from acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes, gestational diabetes, obesity,peripheral arterial disease, cerebrovascular accidents, transientischemic attacks, traumatic brain injuries, amyotrophic lateralsclerosis, burn injuries, sunburn and preeclampsia.
 21. A combinationcomprising a therapeutically effective amount of the polypeptideaccording to claim 1 or an amide, an ester or a salt thereof, and one ormore therapeutically active co-agent.
 22. A combination according toclaim 21 wherein the co-agent is selected from inotropes, betaadrenergic receptor blockers, HMG-Co-A reductase inhibitors, angiotensinII receptor antagonists, angiotensin converting enzyme (ACE) Inhibitors,calcium channel blockers (CCB), endothelin antagonists, renininhibitors, diuretics, ApoA-I mimics, anti-diabetic agents,obesity-reducing agents, aldosterone receptor blockers, endothelinreceptor blockers, aldosterone synthase inhibitors (ASI), a CETPinhibitor, anti-coagulants, relaxin, BNP (nesiritide) and/or a NEPinhibitor.
 23. A pharmaceutical composition comprising a therapeuticallyeffective amount of the polypeptide according to claim 1 or an amide, anester or a salt thereof, and one or more pharmaceutically acceptablecarriers.
 24. A pharmaceutical composition comprising a therapeuticallyeffective amount of the polypeptide according to claim 17, or an amide,an ester or a salt thereof, and one or more pharmaceutically acceptablecarriers.